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https://f1000research.com/articles/8-1769/v1	17 Oct 19	{ "type": "Software Tool Article", "title": "microbiomeDASim: Simulating longitudinal differential abundance for microbiome data", "authors": [ "Justin Williams", "Hector Corrada Bravo", "Jennifer Tom", "Joseph Nathaniel Paulson", "Justin Williams", "Hector Corrada Bravo" ], "abstract": "An increasing emphasis on understanding the dynamics of microbial communities in various settings has led to the proliferation of longitudinal metagenomic sampling studies. Data from whole metagenomic shotgun sequencing and marker-gene survey studies have characteristics that drive novel statistical methodological development for estimating time intervals of differential abundance. In designing a study and the frequency of collection prior to a study, one may wish to model the ability to detect an effect, e.g., there may be issues with respect to cost, ease of access, etc. Additionally, while every study is unique, it is possible that in certain scenarios one statistical framework may be more appropriate than another. Here, we present a simulation paradigm implemented in the R Bioconductor software package microbiomeDASim available at http://bioconductor.org/packages/microbiomeDASim microbiomeDASim. microbiomeDASim allows investigators to simulate longitudinal differential abundant microbiome features with a variety of known functional forms with flexible parameters to control desired signal-to-noise ratio. We present metrics of success results on one particular method called metaSplines.", "keywords": [ "Microbiome", "Differential Abundance", "Longitudinal", "R", "Bioconductor" ], "content": "Introduction\n\nAnalysis of the microbiome aims to characterize the composition and functional potential of microbes in a particular ecosystem. Recent studies have shown the gut microbiome plays an important roles in various diseases, from the efficacy of cancer immunotherapy to the pathogenesis of inflammatory bowel disease (IBD)1–4. While many studies profile static community “snapshots”, microbial communities do not exist within an equilibrium5. To better understand bacterial population dynamics, many studies are expanding to longitudinal sampling and foregoing cross-sectional or single time-point explorations. With a decrease in sequencing costs, more longitudinal data will be generated for varying communities of interest. While data generation will present fewer difficulties, there remain several statistical challenges involved in analyzing these datasets.\n\nThe common approach in the marker-gene survey literature is to perform pairwise differential abundance tests between specific time points and visually confirm, sometimes using smoothing methods like splines, how differences are manifested across time6. These methods require that analysts provide one or more specific time points to test, and the statistical inferences derived from these procedures are specific to these pairwise tests. Other standard methods for longitudinal analysis test for global differences across time, sometimes using non-linear methods including splines to capture dynamic profiles across time7. Incorporating confounding sources of variability, both biological and technical is essential in high-throughput studies8 and require statistical methods capable of estimating both smooth functions and sample-specific characteristics.\n\nSimulating marker-gene amplicon sequencing data presents a variety of challenges related to biological and technical limitations when collecting data. We present a framework for simulating data that can be used across multiple methods for estimating longitudinal differential abundance. This simulation framework allows for appropriate comparison between methods while taking into account some of the unique challenges for the marker-gene amplicon sequencing data, including the following:\n\n1. Non-negative restriction\n\n2. Presence of Missing Data/High Number of Zero Reads\n\n3. Low Number of Repeated Measurements\n\n4. Asynchronous Repeated Measures\n\n5. Small Number of Subjects\n\nThe first two challenges described above are related to the data generating process itself while the following three represent logistical challenges often faced when collecting the data. In microbiomeDASim, we attempt to address these data generating challenges through specific simulation mechanisms described in the Microbiome adaptions section. Similarly, logistical challenges are addressed by allowing users to specify these values flexibly and investigate the corresponding effects, tailoring the simulation to an appropriate setting.\n\nThis package allows investigators to simulate longitudinal differential abundant microbiome features with a variety of known functional forms along with flexible parameters to control design aspects such as signal to noise ratio, correlation structure, and effect size. We highlight the application of a simulation design using one particular method, metaSplines9.\n\n\nMethods\n\nSequencing data are often non-normal. However, transformations, such as log(·) or arcsinh(·), are often applied to raw marker-gene amplicon sequencing data so that the subsequent data is approximately normally distributed. As such, we generate simulated data from a multivariate normal distribution. Using a multivariate normal is a natural choice in this setting as longitudinal correlation structure can be easily incorporated. The following methods focus on cases where the desired microbiome features following appropriate transformation are approximately normally distributed.\n\nAssume that we have data generated from the following distribution,\n\n\n\n\n\nwith Yij representing the ith individual at the jth time point and each individual has qi repeated measurements with i ∈ {1, … , n} and j ∈ {1, … , qi}. We define the total number of observations as N=∑i=1nqi. While this model holds for different choices of qi, throughout this article we will assume, without loss of generality, that the number of repeated measurements is constant, i.e., qi = q ∀ i ∈ {1, … , n}. This means that the total number of observations simplifies to the expression N = qn. Similarly, we split the total patients (n) into two groups, control (n0) and treatment (n1), with the first n0 patients representing the control patients and the remaining n–n0 representing the treatment patients. Subsequently we define the total number of observations in each group as N0 = n0 · q and N1 = n1 · q respectively. Y represents a single taxa/feature to be simulated across the N samples. When simulating multiple features as shown later in the gen_norm_microbiome, these features are assumed to be independent.\n\nPartitioning our observations into control and treatment groups in this way allows us to define the mean vector separately for each group as µ = (µ0,µ1) where µ0 is an N0 × 1 vector and µ1 is an N1 × 1 vector. To generate differential abundance the mean for the control group is held constant µ0 1n0 × 1, but allow the mean vector for the treatment group to vary as a function of time µ1ij (t) = µ0 + f(tj) for i = 1, … , n1 and j = 1, … , q. The form of f(tj) will dictate the functional form of the differential abundance. Note that if f(t1) = 0, then both groups have equal mean at baseline.\n\nWe allow f(tj) to be specified using polynomial basis as\n\n\n\nfor a p dimensional polynomial. We restrict the allowed polynomials to be either linear, p=1, quadratic, p = 2, or cubic, p = 3. For instance, to define a quadratic polynomial one would specify β = (β0, β1, β2)T in the following equation,\n\n\n\nAgain, it is important to note that if β = 0, that the treatment group is assumed to have no differentially abundant timepoints. Typically to simulate no differential abundance, a linear trend is chosen with β0 = β1 = 0.\n\nWhile polynomial functions are often natural choices for longitudinal trends, interest also lies in exploring other non-smooth, i.e., non-differentiable, types of trends. One such form we refer to as oscillating functional forms. These trends include types that transition from linearly increasing to linear decreasing at a point, or vice versa from linearly decreasing to linear increasing. One of the most well known trends of this type is the absolute value function. To allow for flexible choices in oscillating type trends, we allow for these non differentiable linearly connected trends to repeat forming what we call M and W trends. From a biological perspective we could think of these trends as representing spikes in a particular feature that may occur immediately after a treatment dose is given, but then decays rapidly to baseline levels followed by a similar spike and decay upon repeated dosing. These functional trends are operationalized as\n\n\n\nwhere IPk for k = 1, 2, 3 denotes an inflection point where the linear trend changes from increasing to decreasing or vice versa. Note that for these types of trends that the sign of β1 determines whether the trend is initially increasing, i.e. M, (β1 > 0) or initially decreasing, i.e. W, (β1 < 0). By construction, we force the trend line to be exactly zero at IP2 and by doing so the trend is specified completely as β = (β0, β1)T and IP = (IP1, IP2, IP3)T. An implicit restriction on the functional trend is that IP3 ≠ tq. However, we can construct absolute value and inverted absolute value type trends by defining IP1 ∈ (t1, tq) and IP2, IP3 > tq. Again, the key difference for these set of trends is that the inflection points create non-smooth trends.\n\nAn additional extension to linear functional trends is the family of Hockey Stick functional forms. There are two available families of hockey stick functional forms, which are referred to as L_up and L_down within the package. Both of these trends are designed to create two mutually exclusive regions over the time frame specified. These two regions are defined as ℜ1 = (t1, IP) and ℜ2 = (IP, tq) where one of the regions ℜ1 or ℜ2 has linear differential abundance while the other has no differential abundance and IP denotes the inflection point. In the case of the L_up trend, ℜ1 is defined as the non-differentially abundant region and ℜ2 is a linearly increasing region. We can define the functional form as\n\n\n\nNote that with this specification that we do not specify the intercept β0 and instead only need to specify the slope term β1 and the appropriate point of change. We restrict the slope term to be positive, i.e., β1 ∈ (0, ∞) to create the \"up\" trend.\n\nConversely, the L_down trend assumes that ℜ1 is a differentially abundant region that begins with the treatment group higher than the control group and then linearly decreases to the region ℜ2 where there is no differential abundance. We define this functional form as\n\n\n\nNote that in this case we do not specify the point of change directly, but rather it is implicitly implied by the choice of β0 and β1 , i.e. IP = –β0/β1. To ensure that the trend in ℜ1 is properly specified, we place additional restrictions on the parameters so that β0 ∈ (0, ∞) and β1 ∈ (–∞, 0) to ensure the trend is decreasing and check that the choice of β0 and β1 are appropriately defined so that IP ∈ (t1, tq).\n\nExample trends are shown in Figure 1 generated using the mean_trend function.\n\nAs discussed in the Introduction, the multivariate normal is a natural choice for longitudinal simulation due to the ease with which dependency of repeated measures is specified. To encode this longitudinal dependency observations within an individual are assumed to be correlated, i.e. Cor(Yij, Yij') ≠ 0 ∀j ≠ j' and i ∈ {1, … , n}, but observations between individuals are assumed independent, i.e. Cor(Yij, Yi'j) = 0 ∀i ≠ i' and j ∈ {1, … , qi}. To accomplish this we define the block diagonal matrix Σ as Σ = bdiag(Σ1, … , Σn), where each Σi is a q × q covariance matrix for individual i and bdiag(·) indicates that the matrix is block diagonal with all off diagonal elements not in Σi equal to zero. For each individuals covariance matrix, we assume a global standard deviation parameter and correlation component ρ, i.e. Σi = σ2Ω(ρ).\n\nFor instance, if we want to specify an autoregressive correlation structure for individual i the covariance matrix is defined as\n\n\n\nIn this case we are using the first order autoregressive definition and therefore will refer to this as AR(1).\n\nAlternatively, for the compound correlation structure for an individual i' we define the covariance matrix as\n\n\n\nFinally, we allow the user to specify an independent correlation structure for an individual i'' , which assumes that repeated observations are in fact uncorrelated and is defined as\n\n\n\nEach of these correlation structures are referred as AR(1), compound, and independent respectively.\n\nAs discussed in the Introduction, simulating microbiome data presents a variety of unique challenges. In particular there are two data generating restrictions, 1. non-negative restriction and 2. presence of missing data/high number of zero reads, that must be addressed when simulating this data. In this section we will outline some of the specific adaptions of the simulation framework designed to address these issues.\n\n1. Non-negative restriction. One of the most relevant challenges faced with microbiome data, is the restriction of the domain to non-negative values. To assure that the simulated normalized counts are non-negative, one solution is to simply replace the multivariate normal distribution with a multivariate truncated normal distribution. The new data generating distribution is now\n\n\n\nwhere TN indicates the multivariate truncated normal distribution and a is the left-truncation value. To impose zero truncation it is assumed that a = 0. Values from the multivariate truncated normal are drawn using the package tmvtnorm10. Note that the default method for drawing observations from this distribution is rejection sampling which proceeds by first drawing from a multivariate normal and then for all values that fall below a to reject the observed sample and re-sample. This procedure works well when the majority of the distribution falls above the truncation point, but can be computational intensive when the probability of acceptance, pacpt = P(Y > a1N), is low. In our simulation design if the value of µ is sufficiently close to a then rejection sampling is not feasible. In the case there the pacpt ≤ 0.1, non-negative restriction is imposed by censoring negative values and using point imputation with the truncation value a as shown below\n\n\n\nTo remove the non-negative restriction there is an option in the function mvrnorm_sim which can be used to turn-off the domain restriction, but by default the zero truncation is imposed. Note that an alternative option to using the multivariate truncated normal is to use the Johnson translation system which can allow samples to be drawn from a multivariate log normal distribution via an appropriate translation function11. The current implementation uses only the multivariate truncated normal distribution for drawing samples via the zero_trunc option within the mvrnorm_sim() and gen_norm_microbiome() functions.\n\n2. Presence of missing data/high number of zero reads. The second major data generating challenge when simulating microbiome data is the presence of missing data along with a high percentage of features with zero counts. Based on technical limitations when amplifying and sequencing microbiome data, certain features may be present but remain undetected. To approximate this potential for missing features that are truly present, options within mvrnorm_sim allow the user to specify: 1) the percent of individuals to generate missing values from (missing_pct), 2) the number of measurements per individual to assign as missing (missing_per_subject), and 3) the value to impute for missing observations (miss_val). Sample IDs are randomly chosen without replacement across all n units and for each selected ID measurements are randomly selected without replacement from {t2 , … , tq} until the specified number of measurements per individual is achieved. For each missing measurement selected the observed value is replaced with the user specified missing value. Typically the missing value is specified as 0 or as NA with the first case representing a situation where the feature was not included due to technical limitations and the second representing an individual whose data was not collected for a particular time point. The initial value t1 cannot be assigned as missing since it is assumed that all individuals have baseline values collected.\n\nThe current version of the R Bioconductor software package microbiomeDASim12 can be installed in R with the following executable code:\n\n\n\nAlternatively, a development version is available from GitHub and can be accessed at the following repository williazo/microbiomeDASim. The developmental version may contain additional features that are being developed before they are officially introduced into the Biocondutor version. The developmental version can be installed using the following code:\n\n\n\nFor a guided introduction into using the functions see either the package vignette for a static example of how to set up and interact with various options for simulating data or for a dynamic guide see mvrnorm_demo.ipynb, a Jupyter notebook on the GitHub page under the inst/script directory.\n\nmicrobiomeDASim is compatible with major operating systems including Mac OS, Windows and Linux. Package dependencies and system requirements are outlined in the documentation available at GitHub.\n\n\nUse cases\n\nThe primary mechanism for simulating data in the microbiomeDASim package is the function mvrnorm_sim. Through this function, the number of subjects in each group is specified along with the necessary parameters, i.e β, σ2, ρ, and IP, to generate µ and Σ. Below is an example of generating differential abundance using a quadratic trend.\n\n\n\nThe output of the simulation function is a list with 7 total objects. The main object of interest is df, which is a data.frame that contains the complete outcome, Y, IDs for each subject i = 1, … , n, the corresponding time for each observation tj, a group variable indicator, and the outcome with missing data, Y_obs. Both the complete and missing data vectors are also returned as independent objects, Y and Y_obs, respectively, along with the complete mean, µN × 1 = Mu, and covariance matrix, Σ=Sigma. The function also includes a data.frame miss_data which lists any IDs and time points for which missing data was induced. Finally, the function also returns the total number of observations, N=Σi qi. The option dis_plot is used to automatically generate a time-series plot tracking each individuals trajectory along with group mean trajectories. The corresponding plot for this data is shown in Figure 2a.\n\nMissing data in Figure 2b is generated with 20% of subjects randomly selected to have missing values and for each of these subjects to have 2 non-baseline times randomly selected to be missing with the missing observations imputed as 0.\n\nOne important thing to note about the example above is that we generated no missing observations as both missing_pct and missing_per_subject were set to 0. Therefore miss_data was empty. We can compare this to the case below where we induce missingness into the data.\n\n\n\nIn this case we see that for t3 and t5 for subject 10 that our outcome with missing data, Y_obs, is now set as 0 which was specified as our missing value while the complete data has the original value before inducing missingness. The corresponding plot for this simulation with the missing data is shown in Figure 2b.\n\nAs mentioned in the Distributional assumptions section, data are generally generated one feature at a time. However, we may want to simultaneously create data with similar patterns across a number of features with certain features experiencing differential abundance while others have no differential abundance patterns. To do this we can use the function gen_norm_microbiome which lets users specify the number of total features to simulate, features, and the number of total features to be differentially abundant, diff_abun_features. In the example below 10 total features are generated with 4 features having longitudinal differential abundance with an L_down hockey stick type trend.\n\n\n\nThere are two objects returned in this function, bug_feat and Y. The object bug_feat contains all of the sample specific information including Subject ID, timepoint tj, an indicator for group assignment and the Sample_ID which ranges from Sample_1 up to Sample_N. The other object Y is the typical OTU table with rows corresponding to features and column to samples that are commonly used for analysis in packages such as metagenomeSeq13,14.\n\nNext, we want to use our simulation design to test some of the available methods to estimate longitudinal differential abundance. We will examine properties of the estimation method available in the metagenomeSeq14 package to fit a Gaussian smoothing spline ANOVA (SS-ANOVA) model9,15,16 referred to here after as the metaSplines method. We start by generating our simulated data. In this example we will fix parameters so that we have q = 10 repeated measurements on each individual with n0 = n1 = 30 individuals per arm.\n\n\n\nAfter generating the simulated data, we can now create an MRexperiment object needed to fit the model. Note that you can fit either the outcome with the complete data or the outcome with the imputed missing data. In this case we use the complete data.\n\n\n\nNow we can display the estimated interval of differential abundance\n\n\n\nThen we can compare the estimated trend f^(tj) to the truth f(tj) as shown in Figure 3. We observe that the metaSplines estimate falls closely to the true functional form. Further, the confidence intervals for the functional form completely contain the true trend reflecting that the variability in estimation is accurately reflected.\n\nIn the example for metaSplines above we looked at performance using a visual inspection for a single choice of parameter values. Using our simulation framework we can expand our investigation of performance. By knowing the true underlying functional form we can quantify how accurate a particular estimation method captures the truth as a function of sample size per group, number of repeated observations, signal-to-noise strength, type of functional form etc. In order to use the simulated data to compare different longitudinal methods for estimating differential abundance we need to define performance metrics that quantify how accurate an estimate is to the truth. We propose four different performance metrics that can be used when comparing methods.\n\n1. Sensitivity/Specificity ∈ [0, 1]\n\n2. Cosine Similarity f^(t)Tf(t)‖f^(t)‖⋅‖f(t)‖∈[−1,1]\n\n3. Euclidean Distance ‖f^(t)−f(t)‖∈[0,∞]\n\n4. Normalized Euclidean Distance ‖f^(t)‖f^(t)‖−f(t)‖f(t)‖‖∈[0,2]\n\nTo ensure robustness, for each set of parameter values simulated multiple repetitions, B, are required. Sensitivity is defined as the number of repetitions where any differential abundance at any value tj ∊ {t1, . . . , tq} is detected over the total number of repetitions given that the functional form had some true differential abundance over time, i.e. f (tj) ≠ 0 ∀ tj ⇔ µ1 ≠ µ0. Likewise, specificity is defined as the number of repetitions where no differential abundance was detected across all timepoints over the total number of repetitions given that the function form had no true differential abundance over time, i.e., f (tj) = 0 ∀ tj. The other remaining metrics are continuous values that look to compare how closely the estimated mean trend is to the true trend at a set of points tj ∊ {t1, . . . , tq}. Cosine similarity is comparable across different lengths of t, but is not particularly discriminant especially near the boundaries around –1 and 1. The Euclidean distance quantifies how far apart each point is but the length of t is highly influential. Therefore, to make the Euclidean distance comparable across different lengths of repeated observations we can use the normalized Euclidean distance which first transforms the estimated and true functional form into unit vectors and then calculates the distance between these unit vectors.\n\nUsing these performance metrics we simulated data across a range of different parameters settings and then estimated the functional form of the trend using the metaSplines procedure described earlier for a total of 100 repetitions for each parameter setting. Below we show the performance results for a simulation where the functional form was fixed as L_up with an AR(1) correlation structure, ρ = 0.7, and varied the sample size per group, standard deviation, and timepoints from small, medium, and large respectively. The corresponding sensitivity and specificity results are shown in Figure 4a and Figure 4b.\n\nRemaining parameters were varied to create 27 different combinations of repeated measurements, sample size per group, and σ. Points plot are the average result of B = 100 repetitions.\n\nLooking at Figure 4a, in general the sensitivity decreases as σ increases for a fixed sample size and q. For example when n0 = n1 = 10 and q = 6 the estimation procedure is perfectly sensitive (100%) when σ = 1 but has lower sensitivity (42%) when σ = 4. Also as the sample sizes increases for a fixed q and σ, sensitivity generally increases. Likewise, as the number of repeated observations increase, i.e. q increases, the sensitivity increases quite dramatically. This figure suggests that 6 repeated measurements is sufficiently large to detect differential abundance for strong (σ = 1) or medium (σ = 2) signals regardless of the sample size per group. On the other hand, we can look at the specificity in Figure 4b to see that these trends are no longer monotonic. In general we note that as q increases the specificity decreases and that as σ increases the specificity tends to increase. However, the trend for sample size is more nuanced and may variable due to the number of repetitions that were estimable. Using the metaSplines method there were cases with small sample size and repeated observations that the method returned no estimate.\n\nThe sensitivity results shown above were for a single choice of functional form, but this is another potential parameter of interest to test. We ran a similar set of parameter combinations for 7 other functional forms shown in Table 1 below to compare the sensitivity as a function of the type of trend. In this table we can see that the non-differential trends, Oscillating, and variable trends, Hockey Stick, had lower average sensitivity while the linear and quadratic trends tended to perform the best.\n\nNote that the Total Non-Missing Observations is less than the Total Observations.\n\nThe continuous performance metrics for the cosine similarity, Euclidean distance and normalized Euclidean distance are shown in Figure 5 for the L_up trend with AR(1), ρ = 0.7. From this figure we see similar trends as the sensitivity results. Starting from the left most panel we see that the cosine similarity is highest when σ is small, q, n0, n1 are large. The spread of cosine similarity scores when q = 12 are very tightly clustered around 1 while the spread of values when q = 3 or q = 6 is larger. The center plot illustrates that using raw Euclidean distances with a small number of repeated measurements tend to have smaller distances, but this trend is not seen with normalized Euclidean distance in the last panel. Within each value of q in this middle panel there is a consistent trend that as the sample size per group increases the distance generally decreases. Finally moving to the last panel we have the normalized Euclidean distance, which can now be used to compare across different repeated measurement panels. We see a similar trend to the cosine similarity where the distance decreases, meaning better performance, for small σ and large q and n0 = n1.\n\nNote that the red dashed line serves as a reference point at 0.5 and the green dot in each panel represents the mean value across the 100 repetitions.\n\nSimilar to the sensitivity performance metrics shown in Table 1, we can also compare the average value of the continuous performance metrics based on functional form. This is shown in Table 2. Similar trends appear in this table with the linear trends having the highest average cosine similarity scores and lowest average normalized Euclidean distance and non-differentiable trends peforming worse.\n\nNote that the Total Non-Missing Observations is less than the Total Observations.\n\n\nConclusions\n\nWith an increasing emphasis on understanding the dynamics of microbial communities in various settings, longitudinal sampling studies are underway. There remain many statistical challenges when dealing with longitudinal data collected from marker-gene amplicon sequencing. In order to validate and compare methods of estimation for longitudinal differential abundance a unified simulation framework is needed. With the microboimeDASim package the tools are now available to simulate various functional forms for longitudinal differential abundance with added flexibility to control important factors such as the number of repeated measurements per subject, the number of subjects per group, etc. We have shown the benefit of these simulation tools using the metaSplines estimation procedure to compare the performance across a wide range of different parameter settings. In this manner the microbiomeDASim helps meet an important need in the research community to help compare existing methods as well as validate potentially novel methods.\n\n\nData availability\n\nAll data shown from the Use Cases section were simulated and can be generated using source code shown above.\n\n\nSoftware availability\n\nmicrobiomeDASim is available at: http://bioconductor.org/packages/microbiomeDASim.\n\nSource code available from: https://github.com/williazo/microbiomeDASim\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.345856312.\n\nLicense: MIT.", "appendix": "Author contributions\n\n\n\nJW performed analyses, implemented software and wrote first draft of article. HCB contributed to analysis and article review. JT and JNP oversaw analyses and designed experiment.\n\n\nAcknowledgments\n\nAuthors would like to acknowledge Jane Fridlyland and Christina Rabe for helpful discussions and support.\n\n\nReferences\n\nGopalakrishnan V, Spencer CN, Nezi L, et al.: Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients. Science. 2018; 359(6371): 97–103. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRouty B, Le Chatelier E, Derosa L, et al.: Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors. Science. 2018; 359(6371): 91–97. PubMed Abstract \| Publisher Full Text\n\nMatson V, Fessler J, Bao R, et al.: The commensal microbiome is associated with anti-PD-1 efficacy in metastatic melanoma patients. Science. 2018; 359(6371): 104–108. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSivan A, Corrales L, Hubert N, et al.: Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy. Science. 2015; 350(6264): 1084–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYatsunenko T, Rey FE, Manary MJ, et al.: Human gut microbiome viewed across age and geography. Nature. 2012; 486(7402): 222–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKostic AD, Gevers D, Siljander H, et al.: The dynamics of the human infant gut microbiome in development and in progression toward type 1 diabetes. Cell Host Microbe. 2015; 17(2): 260–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMorris A, Paulson JN, Talukder H, et al.: Longitudinal analysis of the lung microbiota of cynomolgous macaques during long-term SHIV infection. Microbiome. 2016; 4(1): 38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeek JT, Scharpf RB, Bravo HC, et al.: Tackling the widespread and critical impact of batch effects in high-throughput data. Nat Rev Genet. 2010; 11(10): 733–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaulson JN, Talukder H, Bravo HC: Longitudinal differential abundance analysis of microbial marker-gene surveys using smoothing splines. bioRxiv. 2017. Publisher Full Text\n\nWilhelm S, Manjunath BG: tmvtnorm: Truncated Multivariate Normal and Student t Distribution. 2015. Reference Source\n\nJohnson NL: Systems of frequency curves generated by methods of translation. Biometrik. 1949; 36(1–2): 149–76. PubMed Abstract \| Publisher Full Text\n\nWilliams J, Bravo HC, Tom J, et al.: williazo/microbiomeDASim: Tools to simulate longitudinal differential abundance for microbiome data (v0.99.2). 2019. http://www.doi.org/10.5281/zenodo.3458563\n\nPaulson JN, Stine OC, Bravo HC, et al.: Differential abundance analysis for microbial marker-gene surveys. Nat Methods. 2013; 10(12): 1200–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaulson JN, Pop M, Bravo HC: metagenomeSeq: Statistical analysis for sparse high-throughput sequncing. Bioconductor package. 2013. Reference Source\n\nGU C: Smoothing spline anova models: R package gss. J Stat Softw. 2014; 58(5): 1–25. Publisher Full Text\n\nGU C: Smoothing spline ANOVA models. Springer, New York, 2nd edition, 2013. Publisher Full Text" }	[ { "id": "55801", "date": "05 Nov 2019", "name": "Leo Lahti", "expertise": [ "Reviewer Expertise Microbiome bioinformatics." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript introduces a new method for simulating longitudinal differential abundance for microbiome data. The method is implemented as an R/Bioc package. The proposed package allows the user to simulate longitudinal microbiome data based on various assumptions, and allows the tuning of key design aspects such as signal-to-noise ratio, correlation structure, effect size and zero inflation. One of the available methods is validated with benchmarking comparisons.\nThe manuscript is technically sound and written in a fluent and easily understandable English. Experiments and statistical analyses have been conducted rigorously. The source code and experiments are openly available via Github but I have not tried to replicate the analysis.\nRealistic simulations are valuable for study design, and help to address questions about sample size, density of time points, experimental costs, etc. The work provides pragmatic solutions to a topical problem in microbiome bioinformatics.\nMajor comments:\nThe simulator provides versatile options to tune signal shape, correlations, and noise. However, I am left wondering how well the simulations correspond to real microbiome data. In particular, it is not clear nor validated how the time series shape and correlation structures correspond to known processes in microbial ecology, such as neutral process, competition models (such as generalized Lotka-Volterra), compositionally aware naive models (Dirichlet-Multinomial), mean-reversing processes (Ornstein-Uhlenbeck). All of these have ecological interpretations and have been visible in recent microbiome time series literature. These models are motivated by known ecological processes, rather than technical modifications on the signal shape; it would be relevant to know how large impact the chosen modeling assumptions might have on the results. Can we expect that the proposed simulator will yield qualitative similar conclusions, even if the connection to ecological mechanisms might be weak?\n\nThe proposed model does not (explicitly) account for heteroschedasticity or overdispersion, and its performance has not been demonstrated with recently popular models of differential abundance, such as DESeq2. It could be true that longitudinal testing of differential abundance requires different methodology. But longitudinal simulators can be also used to simulate cross-sectional data, which is always a snap-shot of longitudinal data. I wonder if the simulator would perform well with standard methods for cross-sectional data; or if it can be shown to yield similar overall distributions. This could provide some additional support for the simulations as the feasibility of the modeling assumptions and their impact on the conclusions remains open.\n\nMinor comments:\nOther simulators for microbiome data and time series are available. One that I am aware of is the seqtime package (https://github.com/hallucigenia-sparsa/seqtime), although that is only available as an R package (and not formally published), but there may be other recent simulators. I did not find other simulation works being cited, it would be good to check if other simulators can be identified in the recent literature, and how they relate to this work.\n\nLack of integration with phyloseq is a weakness, as this class structure is now very popular among the microbiome R users, and many tools build directly on that class structure. It would be useful addition to the package if the simulations could be made available in a phyloseq format.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "5246", "date": "26 Feb 2020", "name": "Justin Williams", "role": "Author Response", "response": "Thank you for your review of the manuscript and suggestions for improvement. Both the manuscript and package have been updated to reflect issues raised above. In the following we address point wise specific comments raised from Version 1 of the manuscript. Major Comments: (1) Thank you for this comment. As an additional step to address the ability of the simulator to reflect real microbiome data we have provided an example of approximating clinical data with longitudinal microbiome data in mice from Turnbaugh et. al, 2009. This section was added to the manuscript under “Approximating Observed Microbiome Data” with further details about how the simulator can be used to complement and expand clinical efforts. In particular, we outline some of the steps to consider when constructing a simulated dataset to approximate a real-world study. Although our simulation design does not explicitly account for ecological processes as mentioned, the focus on the underlying distributional assumption defines the scope of problems which can be addressed. The simulator looks to construct values for a single feature (aggregated at the taxonomic level of interest) and thus does not incorporate correlation between features or compositional constraints. By focusing on only single features of interest we expect that the simulator will yield similar conclusions to those observed in clinical experiments, and thus offers practitioners a useful tool when designing or expanding a longitudinal microbiome study. (2) During the construction of the simulator the variance between both groups is held constant, partly in order to reduce the burden of parameter specification on the user. This choice also reflects a belief that the two groups differ only in their mean trend over time, which is often an appropriate default assumption without particular beliefs about how the heteroskedasticity may differ by group over time. However, it is worthwhile to consider adding a heteroskedastic option to the simulator to incorporate potential differences in noise between groups. While the goal of the simulator focuses on longitudinal designs, it is worthwhile to explore its applicability to cross-sectional data. The simulator function can simulate cross-sectional data by setting num_timepoints=1. Further evaluation of the performance in these cases is merited, but falls outside the scope of this initial software tools manuscript. Minor Comments: (1) We thank the reviewer for pointing to these additional simulator packages. A further investigation of the literature returned multiple packages including seqtime, untb, and WrightFisher with similar goals for simulating longitudinal trends. These packages however focus on simulations from a compositional perspective rather than at a single feature level, and lack some of the documentation and formal publication that accompanies our present package. I have updated the manuscript to include references to these additional packages and note some of the differences in the conclusion. (2) Thank you for this comment. We have added additional conversion functions simulate2MRexperiment and simulate2phyloseq that format simulated data into the respective objects of interest for the metagenomeSeq and phyloseq packages. We have also added details about using these functions within the manuscript." } ] }, { "id": "55802", "date": "06 Nov 2019", "name": "Kris Sankaran", "expertise": [ "Reviewer Expertise statistics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nContributions\nThe authors have developed an R package to simulate longitudinal microbiome time course data, especially where there are difference in trajectories between treatment and control groups. This can be used to address,\nExperimental design: Simulations can guide power analysis, to see whether a proposed study will be well-powered, as a function of assumptions on the generating mechanisms.\n\nMethods comparisons: The effectiveness of different methods will depend on the structure of the data, and simulations provide ground truth from which to make assessments.\n\nThey simulate data one species at a time. Both treatment and control groups are assumed to have gaussian data, truncated below at 0 to reflect transformed counts. Control data are assumed to be drawn from some common mean, but with specified correlation structure over time. Treatment data are assumed to have a mean that deviates from the control according to some function f(), but have the same correlation structure. The authors provide an interface for simulating a few patterns of f() that are believed to be common in real data (e.g., oscillating, quadratic, and linear shapes).\nThe authors share code to display simulated data. They also describe a study evaluating the power of a particular method, 'metaSplines', as simulation parameters are changed.\nEvaluation\nStrengths:\nI like the idea of formalizing simulation-based power analysis. In the microbiome setting, simulations make more sense than theory, but have two issues (1) they are potentially labor-intensive and (2) they can be ad hoc, and never published. By preparing a package, the authors lower the barrier to entry to / introduce a more formal standard for this work, hopefully enabling simulation-based power analysis in the field.\n\nThe paper is generally technically sound, and reads well. Code is available publicly, is clearly documented, and written in a professional style.\n\nWeaknesses:\nThe simulated data are never properly evaluated -- this is my reason for the \"partly\" response in my report. Of course, any simulation is only an approximation of reality, but it would be nice to know along which dimensions the approximation is close, and along which it is poor. This would also set the stage for studying whether the conclusions that you're aiming for (study design or methods choices) are substantially affected by / robust to these deviations in real data. Something in the spirit of graphical inference could be quite interesting here.1\n\nMissed Opportunities:\nThe 'metaSplines' analysis ends somewhat abruptly, because it's not clear what actual conclusions would be drawn from it. I think it would be interesting if you compared another method against it, because you'd be getting at something like the relative efficiency of the approaches (you could also measure their robustness to particular assumptions).\n\nThe functional forms seem somewhat restrictive, though I see their value for people who don't want to spend time writing code. Could you define some kind of interface that makes it easier for people to specify classes of alternatives? E.g., maybe you could let people draw functions interactively, or use as input some examples of microbiome series they see in real data.\n\nDiscussion\nI have trouble believing in any kind of i.i.d. assumption across species. First, the scale of abundance across species tends to differ by orders of magnitude. Second, many species exhibit very similar behavior.\n\nAmong the controls, couldn't some species also vary over time, because of factors in that individual that change which are not specifically treatment?\n\nSetting missing data to 0 is generally bad practice, because then you can't distinguish true zeros from missingness. You should either do proper missing data imputation, or recommend methods that explicitly model the missign values / don't require measurements at equal timepoints.\n\nThe different correlation structures you propose reflect an equispaced sampling design. It wouldn't be too hard to change the correlation structure to allow for unevenly spaced sampling, and it would address your point (4, \"Asynchronous repeated measures\").\n\nCould you create an interactive notebook? E.g., using binder: https://mybinder.org/v2/gh/krisrs1128/microbiome_dasim_example/master. This would make it easier for people (esp. nonexperts) to get acquainted with your work, without having to install jupyter etc.\n\nFor dosage effects, I'd find a (reversed) sawtooth or wavelet-style spike more believable than an oscillating function. But again, this is related to the point of letting people choose their own alternatives.\n\nMinor Comments\nThe caption in Figure 5 seems deprecated.\n\nI don't think you ever defined \"OTU\".\n\nThe library load should say \"microbiome\" not \"microbime\".\n\nThere are still a few typos here and there (e.g., \"differential abundant\" features and \"metrics of success results\"), so I recommend another careful read.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "5247", "date": "26 Feb 2020", "name": "Justin Williams", "role": "Author Response", "response": "Thank you for your careful review of the manuscript and suggestions. Responses to issues raised are shown below for specific points raised. Weakness In the vein of evaluating the robustness of the simulation in approximating reality we have included an additional section “Approximating Observed Microbiome Data” that aims to show how the current package could complement real-world microbiome data. Some of the implications and thought processes for using the simulation package in this setting are discussed within the details of this section. Missed Opportunities We thank the reviewer for this comment. The metaSplines analysis that is included in the manuscript is meant to serve as an illustration of how the simulator could be used to evaluate longitudinal differential abundance methods. In the interest of focusing this software tools manuscript on the simulator package itself, a full comparison of different methods was not investigated. However, this would be a valuable avenue to explore in more depth in a subsequent write-up. Presently we are not aware of any interface within R that would dynamically allow users to draw functions. This would be highly useful and we would like to continue adding in different functional forms within the package. The currently available forms were an initial foray into some potentially relevant types of trends that might be observed. Users with R expertise can modify the mean_trend function to create alternative functional forms, but allowing full user specification may create an unintended burden for many practitioners. In the future, we will consider some alternative options that allow for higher flexibility while maintaining usability. Discussion In our simulation design we are restricting to a single feature of interest when generating data and therefore are inherently ignoring variability across species. This feature simulation can be tailored for individual species of interest and would be run separately in each case. The control group could also vary over time, but from a simulation perspective we are treating the design as if the sample has been norm referenced across time for the control group. Since the main goal of estimation is calculating the difference between the treatment and control group over time, restricting the control group to be invariant over time simplifies the user input and maintains the primary goal of estimation. By default when inducing missingness in the data, the values are treated as NA rather than 0. However, we included the option to specify the value of the missing data to represent cases where there may be some true non-zero occurrence but due to technical limitations such as read depth the values do not appear. The process of generating missingness is meant to align with some of the typical issues such as loss to follow-up when conducting these types of longitudinal designs. Thank you for this comment - as a result we have decided to expand the functionality to allow for asynchronous sampling over a specified interval (using asynch_time=TRUE) or alternatively to have the user specify discrete sampling times for each individual with the mvrnorm_sim_obs function. An example of using each of these asynchronous sampling schemes have been included in the updated manuscript. The compound and independent correlation structures remain unchanged in this unevenly spaced sampling design, but the AR(1) correlation structure now incorporates the amount of time between each sample as \|t_{i}-t_{j}\|. Thank you for this suggestion. The original instructions for installing and running Jupyter with an R kernel were indeed cumbersome. To make the notebook easily interactive, we have re-compiled the materials using Google Colab with a simple badge on top that will allow users to run the code without requiring local installation and setup of Jupyter. Thank you for pointing out these possible functional forms. We will work to expand the functional forms available to include these types of trends in the future. As mentioned earlier the ability to define the mean trend has a natural tradeoff between flexibility and useability. Minor Comments Caption texts, grammatical errors, and typos pointed out have been corrected. Additional read throughs have also been performed to minimize these types of mistakes in the latest draft." } ] } ]	1	https://f1000research.com/articles/8-1769
https://f1000research.com/articles/8-1681/v1	24 Sep 19	{ "type": "Research Article", "title": "Comparison of human population receptive field estimates between scanners and the effect of temporal filtering", "authors": [ "Catherine Morgan", "D. Samuel Schwarzkopf", "D. Samuel Schwarzkopf" ], "abstract": "Background: Population receptive field (pRF) analysis with functional magnetic resonance imaging (fMRI) is an increasingly popular method for mapping visual field representations and estimating the spatial selectivity of voxels in human visual cortex. However, the multitude of experimental setups and processing methods used makes comparisons of results between studies difficult. Methods: Here, we compared pRF maps acquired in the same three individuals using comparable scanning parameters on a 1.5 and a 3 Tesla scanner located in two different countries. We also tested the effect of low-pass filtering of the time series on pRF estimates. Results: As expected, the signal-to-noise ratio for the 3 Tesla data was superior; critically, however, estimates of pRF size and cortical magnification did not reveal any systematic differences between the sites. Unsurprisingly, low-pass filtering enhanced goodness-of-fit, presumably by removing high-frequency noise. However, there was no substantial increase in the number of voxels containing meaningful retinotopic signals after low-pass filtering. Importantly, filtering also increased estimates of pRF size in the early visual areas which could substantially skew interpretations of spatial tuning properties. Conclusion: Our results therefore suggest that pRF estimates are generally comparable between scanners of different field strengths, but temporal filtering should be used with caution.", "keywords": [ "population receptive fields", "site comparison", "replicability", "functional MRI" ], "content": "Introduction\n\nPopulation receptive field analysis with functional magnetic resonance imaging (fMRI) has become a popular method in the toolbox of visual neuroscience. It has not only been used for mapping cortical organization1–3, but also to study spatial integration in the visual cortex4,5, reveal the effects of attention on visual processing6–10, show differences in patients and special populations11–15 and for reconstructing the neural signature of perceptual processes16–20. The most wide-spread technique involves fitting a two-dimensional symmetric Gaussian model of the pRF to the time series of each voxel in visual cortex responding to a set of stimuli1. Estimates of pRF position and size reflect an aggregate of the position preferences and sizes of thousands of neuronal receptive fields of the cells within the imaging voxel, and also incorporates extra-classical receptive field interactions. Further, in fMRI, as underlying neuronal activity is inferred through neurovascular coupling, pRF measurements are affected by hemodynamic factors (although it has been shown that pRFs estimated from fMRI data have a close correspondence with receptive field properties in electrophysiological experiments1,21,22 but see also 23).\n\nThe indirect nature of estimating pRFs from fMRI data suggests therefore that there could be considerable variability in derived measurements. Direct test-retest evaluations with the same experimental setup have shown that pRF mapping experiments are robust and repeatable24–26. However, for different experimental setups, for instance, in terms of the magnetic field strength and the particular pulse sequence used to acquire fMRI data the comparability has not been assessed. The signal-to-noise ratio of MRI is proportional to voxel volume and the strength of the static magnetic field27, and hence pRF measurements at higher magnetic field strength might be more accurate. However, the temporal resolution (or repetition time, TR, of image acquisition), directly affects the contribution of different noise frequencies and the contribution of physiological nuisance factors like respiration or cardiac pulsation. Noise in fMRI data therefore has multiple contributions (physiological, thermal and system related) and the relationship between the temporal signal to noise ratio (TSNR) in a fMRI time course has a non-linear relationship with static SNR28, i.e. gains in static SNR, from for example increased field strength, may not translate to proportional gains in TSNR due to a limit where physiological noise dominates.\n\nHere, we conducted pRF mapping on three individuals using identical TR and voxel size on a 1.5 Tesla (1.5T) scanner in London, United Kingdom and a 3 Tesla (3T) scanner in Auckland, New Zealand. Critically, our aim was not to test the effect of magnetic field strength alone, but rather to compare pRF estimates from typical methods used at each scanning facility that also includes a different field strength. Thus, our findings should reflect a relatively conservative estimate of the variability of pRF parameters under comparable conditions at the two sites.\n\nTypical pRF studies evaluate the goodness-of-fit of the pRF model by means of the coefficient of determination (R2) of the correlation between the observed and predicted time series. This measure, however, strongly depends on the temporal resolution of the signal acquisition. In our recent experiments we have used an accelerated multiband sequence with 1 s TR25,29–32. At this temporal resolution, there is a considerable contribution of high-frequency noise to the signal (Figure 1). Since our analysis does not typically involve any temporal filtering beyond linear detrending (essentially, a wide-bandwidth high-pass filter removing only very low frequencies that are attributed to slow drifts in the signal), this probably explains why the overall R2 in our studies is comparably low compared to those reported by others who use more standard fMRI TRs of 2–3 s. We therefore conducted an analysis comparing pRF parameters obtained using our standard analysis (minimal filtering), to filtering with two low-pass filters that approximate longer TR acquisition.\n\nThe same predicted time series (red curves) is shown overlaid on the unfiltered time series (A), and low-pass filtered time series with kernel 1 s (B) or 2 s (C). R2 is substantially larger for the filtered time series because high-frequency noise has been removed.\n\n\nMethods\n\nWe recruited three healthy adult volunteers (2 female, 1 left-handed, aged 30, 39 and 47) from our pool of repeat participants in functional brain imaging studies in London, United Kingdom. An important criterion for their participation was that they would also visit Auckland, New Zealand, within a few months after the first scan in London. Generally, we could only include participants with normal or corrected-to-normal visual acuity (contact lenses only), and no history of eye disease or neurological, neurodevelopmental or neuropsychiatric disorders. Moreover, they could have no other contraindications to magnetic resonance imaging (e.g. claustrophobia, metal implants). All participants gave written informed consent to take part and henceforth referred to as P1, P2, and P3. All procedures were approved by local ethics review boards at University College London (fMRI/2012/007) and the University of Auckland (017477).\n\nParticipants were scanned twice with a pRF mapping protocol. The first scan took place inside a MAGNETOM Avanto 1.5T MRI scanner (Siemens Healthcare, Erlangen, Germany) at the Birkbeck/UCL Centre for NeuroImaging (BUCNI) in the Experimental Psychology department of University College London, United Kingdom (henceforth referred to as London site). The second scan took place several months later in a MAGNETOM Skyra 3T MRI scanner (Siemens Healthcare, Erlangen, Germany) at the Centre for Advanced Magnetic Resonance Imaging (CAMRI) in the Faculty of Medical & Health Sciences of the University of Auckland, New Zealand (henceforth referred to as Auckland site). In both scans, participants were scanned with six runs for pRF mapping lasting 4 min 20 s each during which functional echo-planar images were acquired. Moreover, at both centres a structural T1 weighted brain image was acquired although the structural image from the second scan (at 3T) was not used in any further analysis. During the scans, participants were instructed to remain as still as possible and fixate continuously on a small dot in the centre of the screen. They were instructed to press a button whenever the fixation dot changed colour.\n\nStimuli were generated and presented using MATLAB (Mathworks; Version R2017a, 9.2.0.538062) and Psychtoolbox (Version 3.0.14)33 at a resolution of 1920 * 1080. At both sites, the screen subtended 34° by 19° of visual angle. Stimuli were presented on a screen at the back of the bore via a mirror mounted on the head coil. At the London site, stimuli were projected onto a screen in the back of the bore while in the Auckland site stimuli were presented on an MRI compatible 32’’ widescreen LCD (Cambridge Research Systems Ltd).\n\nAt both sites, the stimuli generated were matched, and comprised bars containing a dynamic high-contrast ripple pattern as used in previous studies6,13,15,22 on a uniform grey background. Bars traversed the visual field in a regular sequence (e.g. from the bottom to the top), jumping by 0.38° every second. Each sweep of the bar lasted 25 s and so there were 25 jumps of the bar. Each run started with a sweep from the bottom to the top and then the sweep direction was rotated by 45° clockwise on the next sweep. There were thus eight sweeps covering a complete rotation. After the fourth and eighth sweep, a 25 s baseline period (no bars) was presented.\n\nBars were always 0.53° wide and at the longest (when crossing the centre of the visual field) subtended the full screen height, but because they were presented only within a circular region (diameter: 19°, i.e. the height of the screen) they were accordingly shorter at the start and end of each sweep. The outer edge of the ripple stimulus was smoothed by ramping it down to zero over 0.1°. Similarly, the stimulus contrast ramped down to zero from 0.53° to 0.43° eccentricity, thus creating a blank hole around fixation.\n\nA blue dot (diameter: 0.09°) was present in the centre of the screen throughout each scanning run. The whole run was divided into 200 ms epochs. At each epoch, there was a 0.01 probability that the dot could change colour to purple with the constraint that no such colour changes could occur in a row. Participants were instructed to fixate the dot and press a button on a magnetic resonance-compatible button box whenever it changed colour. In addition, a radar screen pattern comprising low-contrast radial and concentric lines around fixation was presented at all times to aid fixation compliance (see 25). The fixation dot and radar screen pattern were also presented during the baseline period.\n\nPrior to each run, we collected 10 dummy volumes to allow ample time for steady-state magnetisation to be reached. During this time, only the fixation dot was presented on a blank grey screen.\n\nAt both sites, we used a 32-channel head coil where we removed the front elements because it impeded the view of the stimulus. This resulted in 20 effective channels covering the back and the sides of the head. Six pRF mapping runs of 260 T2*-weighted image volumes were acquired (including the 10 dummy volumes). We used 36 transverse slices angled to be approximately parallel to the calcarine sulcus (planned using the T1-weighted anatomical image). At both sites, we used an accelerated multiband sequence34,35 at 2.3 mm isotropic voxel resolution, field of view 96x96, and a TR of 1 s. At the London site, the scan had an echo time (TE) of 55 ms, flip angle of 75°, and a multiband/ slice acceleration factor of 4 and rBW 1628 Hz/pixel. At the Auckland site, the scan had a TE of 30 ms, flip angle of 62o, a multiband/slice acceleration factor of 3, an in-plane/parallel imaging acceleration factor of 2 and rBW was 1680 Hz/Px. After acquiring the functional data at the London site, the front portion of the coil was put back on to ensure maximal signal-to-noise levels for collecting a structural scan (a T1-weighted anatomical magnetization-prepared rapid acquisition with gradient echo scan with a 1 mm isotropic voxel size and full brain coverage).\n\nFunctional data were preprocessed in SPM12 (Wellcome Centre for Human NeuroImaging; Version 6906). The first 10 dummy volumes were removed. Then we performed mean bias intensity correction, realignment and unwarping of motion-induced distortions, and coregistration to the structural scan acquired at the London site using default parameters in SPM12. Using FreeSurfer (Version 6.0.0) we further used the structural scan for automatic segmentation and reconstruction as a three-dimensional surface mesh of the pial and grey-white matter boundaries36,37. The grey-white matter surface was then further inflated into a smooth model and a spherical model.\n\nAll further analysis was conducted using our custom SamSrf 6 toolbox38. Functional data were projected from volume space to the surface mesh by finding the nearest voxel located halfway between each vertex in the pial and grey-white matter surface mesh. The time series at each vertex was then linearly detrended and z-standardized before being averaged across the six runs at each scanning site.\n\nFor the temporal filtering analysis, we then further convolved the time series of each vertex with a Gaussian filter with standard deviation of 1 or 2 s, respectively.\n\nWe modelled pRFs as a symmetric, two-dimensional Gaussian defined by x0 and y0, the Cartesian coordinates of the pRF centre in visual space, and the standard deviation of the Gaussian, σ, as a measure of the pRF size. The pRF model predicted the neural response at each TR of the scan by calculating the overlap of the mapping stimulus with this Gaussian pRF profile. A binary mask of 100-by-100 pixels indicated where the stimulus appeared on the screen for each time point. The neural responses were then determined by multiplying each frame of the stimulus mask with the pRF profile and summing over the 10,000 pixels. Subsequently, the time series was convolved with a canonical hemodynamic response function determined from previous empirical data6 and z-standardized.\n\nThe pRF parameters at each vertex were fit using a two-stage procedure. First, we applied a coarse fit which involved an extensive grid search by correlating the actually observed time series against a set of 7650 predicted time series derived from a combination of x0, y0, and σ covering the plausible range for each parameter (see 25,29). The parameters giving rise to the maximal correlation were then retained for the second stage, the fine fit, provided the squared correlation, R2, exceeded 0.01. The fine fit entailed an optimization procedure39,40 to refine the three pRF parameters to further maximize the correlation between observed and predicted time series. Subsequently, we used linear regression between the observed and predicted time series to fit the amplitude, β1, and the baseline intercept, β0. Up until this point, all analyses used raw data without any smoothing or interpolation. However, we then applied a Gaussian smoothing kernel (full width at half maximum = 3 mm) to the final pRF parameter maps on the spherical surface mesh. These smooth maps were used for visualization purposes and delineation of the visual areas. Moreover, they are necessary for calculating the local cortical magnification factor (CMF; Harvey and Dumoulin41) because it requires a smooth visual field map without gaps and scatter. For this we determined the cortical neighbours of each vertex and calculated the area subtended by the polygon formed by their pRF centres. The same procedure was used to determine the cortical surface area (calculation performed by FreeSurfer). To calculate the CMF, we then divided the square root of cortical area by the square root of visual area.\n\nIn addition, for the data comparing the London and Auckland sites we also calculated the noise ceiling as an estimate of the maximum goodness-of-fit that could theoretically be achieved from the data of each voxel. For this, we split the six pRF mapping runs from each site into even and odd numbered runs and averaged them separately. We then calculated the Pearson correlation between these split time series, robs’, and used the Spearman-Brown prophecy formula42,43 to determine, robs, the expected reliability for the average of all six runs:\n\n\n\nThe theoretical maximum observable correlation between the predicted and observed time series, ρo, for each voxel is given by the square root of this correlation44 because\n\n\n\nwhere rpred is the reliability of the predictors, which is 1, and we assume the hypothetical correlation between the observed and predicted time series, ρh, also to be 1. The noise ceiling, the maximum observable goodness-of-fit ρo2 is therefore equal to robs.\n\nWe manually delineated visual areas V1-V4 and V3A using smoothed maps from the London site by determining the borders between regions from the polar angle reversals45 and then also applied these delineations to the maps generated in Auckland. For each region of interest (ROIs) we then extracted pRF data and binned them into eccentricity bands 1° in width, starting from 0.5° and increasing up to 9.5°. For each bin, we then calculated the mean pRF size, and the median CMF, R2, noise ceiling ρo2, or the normalized goodness-of-fit, that is R2 divided by ρo2. To bootstrap the dispersion of these summary statistics, we resampled the data in each bin 1000 times with replacement and determined the central 95% of this bootstrap distribution as a confidence interval. To avoid the inclusion of artifactual data, for pRF size and CMF we only included data from voxels where R2 exceeded 0.15.\n\n\nResults\n\nWe first compared visual field maps from the two sites, London 1.5T and Auckland 3T, by visual inspection. Figure 2 shows polar angle and eccentricity maps of the left hemisphere of one participant from both sites. It is immediately apparent that more voxels survive statistical thresholding (R2>0.1) for the 3T data. The cortical territory occupied by visual field maps is somewhat more extensive and more complete. Nevertheless, the orderly organization of V1-V4, V3A, as well as regions in the LO complex is clearly visible in both scans, and their borders are very similar. We further quantified the map similarity by calculating correlations across all voxels above threshold in both scans (circular correlation for polar angle, Spearman’s ρ correlation for eccentricity). This showed that the polar angle maps were well correlated for two participants (P1: 0.71; P2: 0.71) although the correlation was not as strong in the final participant (P3: 0.44). Eccentricity maps were strongly correlated in all three participants (P1: 0.70; P2: 0.75; P3: 0.67).\n\nPolar angle (A) and eccentricity (B) maps of one participant from the London 1.5T site (left) and the Auckland 3T site (right) displayed on a spherical model of the left hemisphere. Colour wheels denote the pseudo-colour code for visual field maps. No smoothing or interpolation was applied to the mapping data. Voxels were thresholded at R2>0.1. In A, the position of the visual regions have been labelled for reference. The greyscale indicates the cortical folding pattern.\n\nNext, we compared pRF size using 1° wide eccentricity bands to bin the data from each site and then calculating the mean for each bin. We observed that pRF size consistently increased across eccentricities and also along the visual pathway as expected. But crucially, while pRF size varied somewhat between sites, this difference was not systematic across the three participants or the five regions of interest (Figure 3A). In V4 and V3A there was somewhat greater variance at some eccentricities, likely due to the smaller size of these regions compared to V1-V3. Overall, pRF sizes were, however, very similar between sites. Similarly, the median cortical magnification factor (CMF) showed the expected exponential decrease from the central to the peripheral visual field. CMF curves were very comparable for the two sites (Figure 3B), except for somewhat greater CMF at 3T in V1 and V2 in the very central visual field of participants P2 and P3.\n\nMean population receptive field (pRF) size (A) and median cortical magnification factor CMF (B) binned into eccentricity bands for the London 1.5T site (red) and the Auckland 3T site (black). Panels in columns show different visual regions. Panels in rows show the three participants. Error bars denote 95% confidence intervals based on bootstrapping.\n\nWe then repeated this analysis for the goodness-of-fit of the pRF model across eccentricities (Figure 4A). This showed that in V1 and V2 goodness-of-fit was notably greater for the Auckland 3T site than the London 1.5T site in all 3 participants. In higher extrastriate regions, the pattern of results was less clear, although at least for P3 goodness-of-fit was greater also in V3 and V4 (although note that in V4 for P1 very little data with very low model fits was present beyond 6° eccentricity in the Auckland data). In V3A, the model fits at both were similar, but generally lower than in the other regions and with greater variability. However, this was presumably largely driven by the overall signal-to-noise ratio. When we normalized model fits relative to the noise ceiling, ρo2, the maximum goodness-of-fit that could theoretically be achieved given the data from each site, we found no systematic difference in goodness-of-fit between sites (Figure 3B). The curves mostly overlapped for P1 and P2 except in V1 where the London data outperformed the Auckland data. In contrast, for P3 normalized model fits for the Auckland site outperformed the London site in all regions except V3A. The noise ceiling itself was consistently higher for the Auckland data than the London data (Figure 4C).\n\nMedian goodness-of-fit (A), normalized goodness-of-fit (B), and noise ceiling (C) binned into eccentricity bands for the London 1.5T site (red) and the Auckland 3T site (black). Panels in columns show different visual regions. Panels in rows show the three participants. Error bars denote 95% confidence intervals based on bootstrapping.\n\nGoodness-of-fit of the modelled time course derived from pRF estimates and the measured fMRI time course are typically quantified by R2. However, the pRF model does not account for the high frequency noise observed in fast temporal resolution acquisitions like the 1 s TR we used here, which therefore likely results in lower R2 values. Theoretically, high frequency signals could be removed from the data and thus the model fits artifactually improved (Figure 1). We therefore reanalysed the data from the 3T site after temporal low-pass filtering the time series of each voxel by convolving it with a Gaussian kernel of either 1 s or 2 s standard deviation (e.g. Figures 1B and 1C, respectively).\n\nFigure 5 shows visual field maps from the left hemisphere of one participant using the raw data and those after low-pass filtering. A very similar map structure is apparent in all three images. There are, however, also a lot of noise voxels surviving thresholding (R2>0.1) outside the general visual field maps. Conversely, while filtering filled in a few missing voxels within the maps, filtering did not affect the overall structure and did not increase the cortical territory of the responsive region.\n\nColour wheels denote the pseudo-colour code for visual field maps. No smoothing or interpolation was applied to the mapping data. Voxels were thresholded at R2>0.1. The position of the visual regions have been labelled for reference. The greyscale indicates the cortical folding pattern. A. Unfiltered data. B. Low-pass filtered with kernel 1 s. C. Low-pass filtered with kernel 2 s.\n\nNext, we quantified mean pRF size and compared this for the three data sets (Figure 6A). In the early areas V1-V3, pRF size is consistently greater for the filtered time series, especially at the longer kernel of 2 s. In V4 and V3A, pRF size remained relatively similar between conditions. We also quantified the median goodness-of-fit of the pRF model (Figure 6B) and observed consistently greater model fits for both the low-pass filtered time series in V1 to V4. A similar pattern could be seen in V3A but results were generally more variable, especially in P1.\n\nMean population receptive field (pRF) size (A) and median goodness-of-fit (B) binned into eccentricity bands for the Auckland data without filtering (black), and low-pass filtering with kernel 1 s (blue) and 2 s (green). Panels in columns show different visual regions. Panels in rows show the three participants. Error bars denote 95% confidence intervals based on bootstrapping.\n\nRaw results are available from Open Science Framework as Underlying data46.\n\nHere, we compared pRF mapping data acquired using the same stimuli and participants and under comparable conditions at two sites, a 1.5T scanner in London, United Kingdom, and a 3T scanner (by the same vendor) in Auckland, New Zealand. Generally, pRF model fits were better in the Auckland data, probably in large part because of the approximately double signal-to-noise ratio from the greater static magnetic field. However, despite this difference in accuracy of data fitting, we found that visual field maps and actual estimates of pRF size and local cortical magnification were very similar between the two sites.\n\nAlthough the London data were always acquired first, this is unlikely to explain our findings. All our participants were familiar with the fMRI environment, including undertaking previous pRF mapping studies. Therefore, it is unlikely that there should have been substantial training effects that could for example have changed the amount of head motion or fixation compliance between sessions. The exact parameters of the pulse sequences used and general differences in the image quality of the two scanners could of course also be contributing factors to inter-site differences. We did not apply any correction of distortions caused by the static magnetic field47 to either site data. Moreover, the exact slice positioning, and thus how the voxel grid resolved the grey matter, may also have differed between sites. Relevant to this, in our previous work quantifying the test-retest reliability of pRF maps we found that reliability was greater for scanning sessions in close succession on the same day than for sessions on different days25. This could have been caused by fluctuations in the scanner hardware itself but also relate to differences in head position at set up. While in both cases participants were removed from the scanner between the repeat sessions, it is highly likely that positioning was more similar for the sessions conducted on the same day.\n\nWhile we took painstaking steps to match the visual displays at the two sites as closely as possible, due to constraints of the experimental setup there may have been small differences between the stimuli presented. In London, images were projected onto a screen and this necessitated focussing and scaling the projected image to be of the exact size. The image in London may have been somewhat blurrier and the viewing angle more variable than in Auckland where we used a clear liquid crystal display that was always placed at the exact same position at the back of the bore. Naturally, the exact viewing angle of the stimuli also depends on the viewing distance and there may have been subtle variation between the sites although this is unlikely to have produced any systematic differences.\n\nNevertheless, the general extent of the visually responsive area of cortex containing clear retinotopic maps was very comparable across the sites, as were estimates of spatial tuning and cortical magnification. Therefore, we conclude that pRF estimates are very robust across scanning sites, even when using different magnetic field strengths.\n\nAt the short TR of 1 s as used in our study, noise contributes high-frequency signals to the fMRI response that are irrelevant to the sluggish blood oxygen level dependent activity the pRF model seeks to characterize. This explains why the model fits in most of our studies are relatively low compared to those reported in the literature using more conventional TRs. We therefore also sought to test what effect temporal low-pass filtering had on pRF parameter estimates. Unsurprisingly, low-pass filtering enhanced the goodness-of-fit of pRF models. However, while this filled in a few gaps in the maps it largely boosted the number of noise voxels outside the visually responsive cortex.\n\nCrucially, low-pass filtering also generally enlarged estimates of pRF size in the early visual regions V1-V3 while data in higher regions were mostly unaffected. The pRF size estimates for the unfiltered data accord well with previous research and what one would expect from electrophysiological recordings1,21,22,41. Therefore, the pRF sizes of the filtered data presumably reflect an overestimate that seems unrealistically high (e.g. for P2 mean pRF size in central V1 was close to 2° for the most heavily filtered data).\n\nThe reason why filtering increased pRF sizes in early areas is probably the fact that filtering blurred together signals from stimuli presented close in time (proximal bar positions were presented only a few seconds apart). In the early regions where pRFs are small this would therefore be modelled as a larger pRF, whereas in higher areas in which pRFs are large enough to encompass adjacent bar positions this should not affect estimates of pRF size. A slower stimulus design where each bar position is stimulated for 2-3 s, or a random instead of an ordered stimulus sequence, could probably counteract this problem but this would come at the expense of longer scanning durations. In any case, for our standard design using 1 s TR low-pass filtering clearly has no practical advantages because it does not appear to fundamentally improve map quality and skews pRF size estimates in the early visual areas.\n\nSince we only tested three participants in this study, our analyses were not designed to detect subtle differences between the scanner environments or small effects of temporal filtering. Retinotopic maps and pRF data are highly replicable25 and can also be used for case studies on individuals. We were therefore most interested in how consistent results are across all three participants. Our descriptive analysis of the data included bootstrapped confidence intervals, which permit an inference about the statistical evidence in each participant, and thus each individual participant essentially constitutes a replication.\n\nIn summary, our results show that pRF mapping with fast 1 s TR produces similar and reliable results on different scanners, even using different magnetic field strengths. It would be interesting to conduct a similar comparison between 3T and 7T scans as these are becoming increasingly commonplace.\n\nOpen Science Framework: pRF data comparison between London 1.5T & Auckland 3T. https://doi.org/10.17605/OSF.IO/WX4HP46.\n\nThis project contains Lon-Akl.zip, which itself contains underlying data for each participant in folders 30fl, 39mr and 47fr. These files each contain the following underlying data:\n\nanatomy (the model of the subject's brain anatomy).\n\nprf_bucni (functional MRI data (pRF maps) from London).\n\nprf_camri (functional MRI data (pRF maps) from Auckland).\n\nROIs (delineation (FreeSurfer label files) for the visual areas).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe would like to thank the staff of the Centre for Advanced Magnetic Resonance Imaging (CAMRI) and the Birkbeck-UCL Centre for NeuroImaging (BUCNI) for technical support and the Centre for eResearch, University of Auckland, for computing support.\n\n\nReferences\n\nDumoulin SO, Wandell BA: Population receptive field estimates in human visual cortex. NeuroImage. 2008; 39(2): 647–660. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmano K, Wandell BA, Dumoulin SO: Visual field maps, population receptive field sizes, and visual field coverage in the human MT+ complex. J Neurophysiol. 2009; 102(5): 2704–2718. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWinawer J, Horiguchi H, Sayres RA, et al.: Mapping hV4 and ventral occipital cortex: the venous eclipse. J Vis. 2010; 10(5): 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDumoulin SO, Hess RF, May KA, et al.: Contour extracting networks in early extrastriate cortex. J Vis. 2014; 14(5): 18. PubMed Abstract \| Publisher Full Text\n\nHarvey BM, Dumoulin SO: Visual motion transforms visual space representations similarly throughout the human visual hierarchy. NeuroImage. 2016; 127: 173–185. PubMed Abstract \| Publisher Full Text\n\nde Haas B, Schwarzkopf DS, Anderson EJ, et al.: Perceptual load affects spatial tuning of neuronal populations in human early visual cortex. Curr Biol. 2014; 24(2): R66–67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKlein BP, Harvey BM, Dumoulin SO: Attraction of position preference by spatial attention throughout human visual cortex. Neuron. 2014; 84(1): 227–237. PubMed Abstract \| Publisher Full Text\n\nKlein BP, Fracasso A, van Dijk JA, et al.: Cortical depth dependent population receptive field attraction by spatial attention in human V1. NeuroImage. 2018; 176: 301–312. PubMed Abstract \| Publisher Full Text\n\nKay KN, Weiner KS, Grill-Spector K: Attention reduces spatial uncertainty in human ventral temporal cortex. Curr Biol. 2015; 25(5): 595–600. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVo VA, Sprague TC, Serences JT: Spatial Tuning Shifts Increase the Discriminability and Fidelity of Population Codes in Visual Cortex. J Neurosci. 2017; 37(12): 3386–3401. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLevin N, Dumoulin SO, Winawer J, et al.: Cortical maps and white matter tracts following long period of visual deprivation and retinal image restoration. Neuron. 2010; 65(1): 21–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoffmann MB, Kaule FR, Levin N, et al.: Plasticity and stability of the visual system in human achiasma. Neuron. 2012; 75(3): 393–401. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchwarzkopf DS, Anderson EJ, de Haas B, et al.: Larger extrastriate population receptive fields in autism spectrum disorders. J Neurosci. 2014; 34(7): 2713–2724. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClavagnier S, Dumoulin SO, Hess RF: Is the Cortical Deficit in Amblyopia Due to Reduced Cortical Magnification, Loss of Neural Resolution, or Neural Disorganization? J Neurosci. 2015; 35(44): 14740–14755. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnderson EJ, Tibber MS, Schwarzkopf DS, et al.: Visual Population Receptive Fields in People with Schizophrenia Have Reduced Inhibitory Surrounds. J Neurosci. 2017; 37(6): 1546–1556. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKok P, de Lange FP: Shape perception simultaneously up- and downregulates neural activity in the primary visual cortex. Curr Biol. 2014; 24(13): 1531–1535. PubMed Abstract \| Publisher Full Text\n\nKok P, Bains LJ, van Mourik T, et al.: Selective Activation of the Deep Layers of the Human Primary Visual Cortex by Top-Down Feedback. Curr Biol. 2016; 26(3): 371–376. PubMed Abstract \| Publisher Full Text\n\nKok P, van Lieshout LL, de Lange FP: Local expectation violations result in global activity gain in primary visual cortex. Sci Rep. 2016; 6: 37706. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEkman M, Kok P, de Lange FP: Time-compressed preplay of anticipated events in human primary visual cortex. Nat Commun. 2017; 8: 15276. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSenden M, Emmerling TC, van Hoof R, et al.: Reconstructing imagined letters from early visual cortex reveals tight topographic correspondence between visual mental imagery and perception. Brain Struct Funct. 2019; 224(3): 1167–1183. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWinawer J, Rauschecker AM, Parvizi J, et al.: Population receptive fields in human visual cortex measured with subdural electrodes. J Vis. 2011; 11: 1196. Publisher Full Text\n\nAlvarez I, de Haas BA, Clark CA, et al.: Comparing different stimulus configurations for population receptive field mapping in human fMRI. Front Hum Neurosci. 2015; 9: 96. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKeliris GA, Li Q, Papanikolaou A, et al.: Estimating average single-neuron visual receptive field sizes by fMRI. Proc Natl Acad Sci U S A. 2019; 116(13): 6425–6434. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSenden M, Reithler J, Gijsen S, et al.: Evaluating population receptive field estimation frameworks in terms of robustness and reproducibility. PLoS One. 2014; 9(12): e114054. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Dijk JA, de Haas B, Moutsiana C, et al.: Intersession reliability of population receptive field estimates. NeuroImage. 2016; 143: 293–303. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBenson NC, Jamison KW, Arcaro MJ, et al.: The Human Connectome Project 7 Tesla retinotopy dataset: Description and population receptive field analysis. J Vis. 2018; 18(13): 23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEdelstein WA, Glover GH, Hardy CJ, et al.: The intrinsic signal-to-noise ratio in NMR imaging. Magn Reson Med. 1986; 3(4): 604–618. PubMed Abstract \| Publisher Full Text\n\nMurphy K, Bodurka J, Bandettini PA: How long to scan? The relationship between fMRI temporal signal to noise ratio and necessary scan duration. NeuroImage. 2007; 34(2): 565–574. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoutsiana C, de Haas B, Papageorgiou A, et al.: Cortical idiosyncrasies predict the perception of object size. Nat Commun. 2016; 7: 12110. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoutsiana C, Soliman R, de Wit L, et al.: Unexplained Progressive Visual Field Loss in the Presence of Normal Retinotopic Maps. Front Psychol. 2018; 9: 1722. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Haas B, Schwarzkopf DS: Spatially selective responses to Kanizsa and occlusion stimuli in human visual cortex. Sci Rep. 2018; 8(1): 611. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHughes AE, Greenwood JA, Finlayson NJ, et al.: Population receptive field estimates for motion-defined stimuli. NeuroImage. 2019; 199: 245–260. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrainard DH: The Psychophysics Toolbox. Spat Vis. 1997; 10(4): 433–6. PubMed Abstract \| Publisher Full Text\n\nBreuer FA, Blaimer M, Heidemann RM, et al.: Controlled aliasing in parallel imaging results in higher acceleration (CAIPIRINHA) for multi-slice imaging. Magn Reson Med. 2005; 53(3): 684–691. PubMed Abstract \| Publisher Full Text\n\nMoeller S, Yacoub E, Olman CA, et al.: Multiband multislice GE-EPI at 7 tesla, with 16-fold acceleration using partial parallel imaging with application to high spatial and temporal whole-brain fMRI. Magn Reson Med. 2010; 63(5): 1144–1153. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDale AM, Fischl B, Sereno MI: Cortical surface-based analysis. I. Segmentation and surface reconstruction. NeuroImage. 1999; 9(2): 179–194. PubMed Abstract \| Publisher Full Text\n\nFischl B, Sereno MI, Dale AM: Cortical surface-based analysis. II: Inflation, flattening, and a surface-based coordinate system. NeuroImage. 1999; 9(2): 195–207. PubMed Abstract \| Publisher Full Text\n\nSchwarzkopf DS, de Haas B, Alvarez I: SamSrf 6 - Toolbox for pRF modelling. Open Science Framework. 2018. Publisher Full Text\n\nNelder JA, Mead R: A Simplex Method for Function Minimization. Comput J. 1965; 7(4): 308–313. Publisher Full Text\n\nLagarias J, Reeds J, Wright M, et al.: Convergence properties of the Nelder—Mead simplex method in low dimensions. SIAM J Optim. 1998; 9(1): 112–147. Publisher Full Text\n\nHarvey BM, Dumoulin SO: The relationship between cortical magnification factor and population receptive field size in human visual cortex: constancies in cortical architecture. J Neurosci. 2011; 31(38): 13604–13612. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrown W: Some Experimental Results in the Correlation of Mental Abilities1. Brit J Psychol. 1910; 3(3): 296–322. Publisher Full Text\n\nSpearman C: Correlation Calculated from Faulty Data. Brit J Psychol. 1910; 3(3): 271–295. Publisher Full Text\n\nSpearman C: The Proof and Measurement of Association between Two Things. Am J Psychol. 1904; 15(1): 72–101. Publisher Full Text\n\nSereno MI, Dale AM, Reppas JB, et al.: Borders of multiple visual areas in humans revealed by functional magnetic resonance imaging. Science. 1995; 268(5212): 889–893. PubMed Abstract \| Publisher Full Text\n\nSchwarzkopf D, Morgan C: pRF data comparison between London 1.5T & Auckland 3T. 2019. http://www.doi.org/10.17605/OSF.IO/WX4HP\n\nHutton C, Bork A, Josephs O, et al.: Image distortion correction in fMRI: A quantitative evaluation. NeuroImage. 2002; 16(1): 217–240. PubMed Abstract \| Publisher Full Text" }	[ { "id": "54262", "date": "25 Oct 2019", "name": "Noah Benson", "expertise": [ "Reviewer Expertise Data science / visual neuroscience" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPopulation receptive field (pRF) modeling is an incredibly important and common technique in visual and perceptual neuroscience. PRF mapping is also complex, and, despite its popularity, few examinations of how the many incidental complexities of collecting pRF maps affect the maps themselves have been reported. This lack makes comparison and interpretation of pRF results across sites and studies difficult. In this article, Morgan and Schwarzkopf provide a straightforward examination of two of these incidental complexities of retinotopic pRF mapping: the field-strength of the scanner and the choice to low-pass filter the BOLD time-series. Encouragingly, they find that the effect of the field-strength is minimal; however, the effects of the filtering are non-trivial.\nI have no major concerns regarding the methods of this paper, but I do have a few suggestions. First, it is not clear if the authors used the same hemodynamic response function (HRF) for all subjects or if a single canonical function was fit to empirical data from each subject individually, nor do the authors explain which canonical HRF was used; I would like to see the HRF form(s) reported explicitly. Secondly, the use of linearly-spaced eccentricity bands for the various further analyses is a somewhat questionable choice given that the number of vertices between 8.5° and 9.5° will be substantially smaller than the number of vertices between 0.5° and 1.5°. This effect may partly explain why the errors in some figures (e.g. Figure 6) are quite large at higher eccentricities. A principled way to obtain similarly-sized eccentricity bins might be to use Horton and Hoyt's 1991 equation 1 for cortical magnification [CMF(e) = (17.3 / (0.75 + e))2 (mm/deg)2 for eccentricity e], integrate it over the visual field to obtain an estimate of cortical surface area in terms of eccentricity [SAF(e) (mm2)], then to invert this [SAF-1(a) (deg) for cortical surface area a] and choose eccentricity values that correspond to values of SAF-1(a) for linearly-spaced surface-area values of a.\nDespite the above suggestions, the results of the paper are quite clear: (1) pRF models are robust to changes in the field strength of the scanner, and (2) temporal filtering inflates the R2 and size parameter of the pRF fits without substantially changing the topographic structure of the retinotopic maps. Both of these insights are useful additions to the field, especially given the rapid rate of change in MRI technology and the frequency with which temporal smoothing is applied to pRF mapping data. More work is needed throughout the field to quantify these kinds of effects, and this paper is a useful contribution to that effort. One minor suggestion is that the authors might explicitly state in the discussion that the two scanners share a manufacturer; this is both a limitation of the comparison, in that the results may not generalize across manufacturers, and one of its strengths, in that one can be more confident of the findings with respect to field-strength because the manufacturer was not an additional variable across sites.\nMy biggest concerns about the paper center on the supplemental data. Although the authors have provided enough data to reproduce the figures and aspects of the study, the data provided are incomplete and fail to conform to contemporary standards of reusability and interoperability. For one, all of the supplemental data files could have been provided in well established community-vetted formats (such as NIFTI files), yet none of the data are provided in such formats (in fact many of the files cannot be easily read without an expensive MATLAB license). Additionally, only some of the data are provided: FreeSurfer reconstructions, anonymized anatomical images, and various raw data are not provided. These data really ought to be part of the dataset, and ideally should be provided in accordance with the BIDS spec 2. A few examples of things that cannot be examined with the current dataset: concerns about field distortions, questions about partial voluming artifacts, concerns about signal attenuation near vessels, questions about how the results relate to the surface area of the visual areas, and questions about how the results generalize to frontal cortex. To be clear, none of these examples are likely to impact the conclusions of the study, but I point them out as examples of how the current dataset fails to meet the evolving standards of data-availability in the field.\nBeyond these comments, I have only minor concerns that are largely aesthetic in nature: The use of the prime symbol (′) and the single quote (') is inconsistent and, due to the occasional adjacency of variable symbols with subscripts next to commas, can be a bit confusing. Figures 3, 4, and 6 contain a lot of information, and I feel that a panel that summarizes or combines the data across subjects in some way would be useful and easier to interpret. The eccentricity colormap used in Figure 2 is circular, which causes some ambiguity between very different values. Figure 1 seems to have a larger range (along the y axis) for the filtered data than for the unfiltered data. It is not clear to me how the filtering described would produce a signal with nearly twice the range of values as the original signal. This might be a plotting error.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5235", "date": "26 Feb 2020", "name": "Catherine Morgan", "role": "Author Response", "response": "We’d like to thank the reviewer for their considered questions and useful suggestions on this manuscript. In our response we have reproduced the report, and commented immediately after each point. “Population receptive field (pRF) modeling is an incredibly important and common technique in visual and perceptual neuroscience. PRF mapping is also complex, and, despite its popularity, few examinations of how the many incidental complexities of collecting pRF maps affect the maps themselves have been reported. This lack makes comparison and interpretation of pRF results across sites and studies difficult. In this article, Morgan and Schwarzkopf provide a straightforward examination of two of these incidental complexities of retinotopic pRF mapping: the field-strength of the scanner and the choice to low-pass filter the BOLD time-series. Encouragingly, they find that the effect of the field-strength is minimal; however, the effects of the filtering are non-trivial. I have no major concerns regarding the methods of this paper, but I do have a few suggestions. First, it is not clear if the authors used the same hemodynamic response function (HRF) for all subjects or if a single canonical function was fit to empirical data from each subject individually, nor do the authors explain which canonical HRF was used; I would like to see the HRF form(s) reported explicitly.” >> For all subjects we used the same canonical HRF based on previous data (de Haas et al. 2014). The reviewer is right that the HRF used can have an effect on the obtained pRF parameters but this effect is typically very small (Dumoulin & Wandell, 2008). In our previous study quantifying the test-retest reliability of pRF parameters on the same scanner we found only minimal effects of obtaining subject-specific HRF fits. While it is indeed possible that the HRF differs between our scanners in the present study, this effect is also unlikely to alter our conclusions considering the already very similar pRF parameters we observed. We thank you for pointing this ambiguity out and will edit the manuscript accordingly. “Secondly, the use of linearly-spaced eccentricity bands for the various further analyses is a somewhat questionable choice given that the number of vertices between 8.5° and 9.5° will be substantially smaller than the number of vertices between 0.5° and 1.5°. This effect may partly explain why the errors in some figures (e.g. Figure 6) are quite large at higher eccentricities. A principled way to obtain similarly-sized eccentricity bins might be to use Horton and Hoyt's 1991 equation 1 for cortical magnification [CMF(e) = (17.3 / (0.75 + e))2 (mm/deg)2 for eccentricity e], integrate it over the visual field to obtain an estimate of cortical surface area in terms of eccentricity [SAF(e) (mm2)], then to invert this [SAF-1(a) (deg) for cortical surface area a] and choose eccentricity values that correspond to values of SAF-1(a) for linearly-spaced surface-area values of a.” >> The reviewer raises a very important point and we have previously debated this in the lab and also with other researchers. Linearly scaled eccentricity bands is the standard procedure used by most pRF studies . It is true that a logarithmic scaling of eccentricity bands would ensure approximately equal amounts of data per eccentricity band, and thus ensure relatively even error margins. It however seems to us unlikely that this will alter our conclusions in any way or that this will reveal additional information. Practically, it would reduce the number of bins which could also be suboptimal for comparing the findings by visual inspection. As an example, we included the equivalent to the figure for the pRF size from the two scanning sites but using logarithmic bands online: https://osf.io/nyj4z/ To our eyes this does not alter what we can interpret from this plot. Note also that the most peripheral band still contains less data because edge artifacts are not always consistent and thus we cannot ensure equal surface area for this band. We therefore decided to go with tradition and stick with linear bins. “Despite the above suggestions, the results of the paper are quite clear: (1) pRF models are robust to changes in the field strength of the scanner, and (2) temporal filtering inflates the R2 and size parameter of the pRF fits without substantially changing the topographic structure of the retinotopic maps. Both of these insights are useful additions to the field, especially given the rapid rate of change in MRI technology and the frequency with which temporal smoothing is applied to pRF mapping data. More work is needed throughout the field to quantify these kinds of effects, and this paper is a useful contribution to that effort. One minor suggestion is that the authors might explicitly state in the discussion that the two scanners share a manufacturer; this is both a limitation of the comparison, in that the results may not generalize across manufacturers, and one of its strengths, in that one can be more confident of the findings with respect to field-strength because the manufacturer was not an additional variable across sites.” >> We’d like to thank the reviewer for highlighting this very important point and will add it to the discussion. “My biggest concerns about the paper center on the supplemental data. Although the authors have provided enough data to reproduce the figures and aspects of the study, the data provided are incomplete and fail to conform to contemporary standards of reusability and interoperability. For one, all of the supplemental data files could have been provided in well established community-vetted formats (such as NIFTI files), yet none of the data are provided in such formats (in fact many of the files cannot be easily read without an expensive MATLAB license). Additionally, only some of the data are provided: FreeSurfer reconstructions, anonymized anatomical images, and various raw data are not provided. These data really ought to be part of the dataset, and ideally should be provided in accordance with the BIDS spec 2. A few examples of things that cannot be examined with the current dataset: concerns about field distortions, questions about partial voluming artifacts, concerns about signal attenuation near vessels, questions about how the results relate to the surface area of the visual areas, and questions about how the results generalize to frontal cortex. To be clear, none of these examples are likely to impact the conclusions of the study, but I point them out as examples of how the current dataset fails to meet the evolving standards of data-availability in the field.” >> We are very supportive of the idea of open science in general, for data integrity and reproducibility. However, data transparency must be traded off against ethical and data protection concerns. Any brain imaging data is by nature not fully de-identifiable, especially if it contains anatomical/morphological information. This is a particular issue with a small sample size like this (which is not uncommon for retinotopic mapping studies). Moreover we also have reason to believe that under New Zealand laws (specifically the Privacy Act (1994) and the Health Information Privacy Code (HIPC); a set of legally-binding rules for applying this act) we cannot share our full imaging data (including volumetric EPI data and FreeSurfer reconstructions) publicly without any custodianship as it can only be shared for research and for the same purpose as our original ethical approval. We are currently in the process of seeking legal guidance on this point and determine a consistent policy on data sharing within our institution. However, even in the absence of all the raw data, the data we do share are fully transparent and theoretically permit anyone to reproduce our findings. As opposed to the reviewer’s concern, our data do not require a MATLAB license but can be read using the free software Octave and FreeSurfer. Octave misses some of the functionality of MATLAB and we are currently working on opening this up but critically the data are fully accessible. All the pre-processed time series for individual voxels/vertices are also included which would allow an interested research to carry out their own pRF analysis on these data. Regarding the format, at present we always share our data in the MATLAB format because that is what our SamSrf toolbox uses. It is convenient because it allows us to store all the functional data per hemisphere in one file, in a relatively straightforward data structure, with maps and time series information of all vertices of the cortical reconstruction in a single 2D matrix. We however see the merit in a ready exchange of data between different platforms and are currently considering and debating adding support for standard formats that allow this, but in our view this is outside the scope of this revision.. “Beyond these comments, I have only minor concerns that are largely aesthetic in nature: The use of the prime symbol (′) and the single quote (') is inconsistent and, due to the occasional adjacency of variable symbols with subscripts next to commas, can be a bit confusing.” >> We thank the reviewer for pointing this out and will change this in our revision. “Figures 3, 4, and 6 contain a lot of information, and I feel that a panel that summarizes or combines the data across subjects in some way would be useful and easier to interpret.” >> This is an interesting point. Considering our sample size is 3 we believe presenting individual data is important. However, the group results are also very clear and we therefore decided to present only group averages in the main manuscript now, thank you for the suggestion, and instead relegate the individual plots to the Supplementary Information. “The eccentricity colormap used in Figure 2 is circular, which causes some ambiguity between very different values.” >> The choice of the same colour scheme for polar angle and eccentricity is in part a leftover from old days in which eccentricity maps were based on phase-encoded analysis. We agree that this could be somewhat confusing, although our rendering ensures that this is not the case. The fovea is represented by red whereas the most peripheral eccentricities are shown in purple. The colour scheme does not cycle back. Instead, all eccentricities above the outer edge of the stimulus (9.5 degrees) are set to purple. “Figure 1 seems to have a larger range (along the y axis) for the filtered data than for the unfiltered data. It is not clear to me how the filtering described would produce a signal with nearly twice the range of values as the original signal. This might be a plotting error.” >> We thank the reviewer for pointing this out. This is an artifact in this illustration and the Y-axis should really be in arbitrary units. We will change this in our revision. >> Response References de Haas B, Schwarzkopf DS, Anderson EJ, et al.: Perceptual load affects spatial tuning of neuronal populations in human early visual cortex. Curr Biol. 2014; 24(2): R66–67. Dumoulin SO, Wandell BA: Population receptive eld estimates in human visual cortex. NeuroImage. 2008; 39(2): 647–660." } ] }, { "id": "56563", "date": "12 Dec 2019", "name": "Paola Binda", "expertise": [ "Reviewer Expertise Physiology of the visual system" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\npRF mapping provides a quantitative description of cortical representations, including the spatial representation in visual cortical areas. While pRF parameters are extensively used to quantitatively compare across subjects and conditions, we are well aware that such parameters depend on both neural factors and on nuisance variables that are related to the dynamics of BOLD. However, we lack a precise understanding of how such nuisance variables might affect each of the pRF parameters. Morgan & Schwarzkopf make an important effort in this direction and test the impact of static SNR (affected by MR field strength) and temporal SNR (affected by temporal filtering) on spatial pRF parameters in visual cortical areas.\nI have no major concerns with the study and only offer few suggestions that hopefully will increase the usability of the reported results:\nIt would be useful to explicitly describe one (or more) models that predict changes of pRF parameters with SNR.\nI feel one intuitive (and probably wrong) model invokes a non-linear thresholding effect. The logic is, approximately, as follows: when BOLD responses are stronger, each voxel responds to a larger range of stimulus position and the pRF size is consequently increased. Is this one of the hypotheses that the study intends to test (and falsify)?\nThis particular model would predict that beta values (indexing BOLD response amplitude) and pRF size estimates correlate positively. Since both indices were collected here, could the authors measure this (lack of) correlation?\nIn addition, this model would incorrectly predict larger pRF size estimates at higher MR fields, under the assumption that response amplitude is positively related to SNR. Could the authors report whether this assumption (larger beta-values in the higher SNR, i.e. 3T, dataset) is correct for their specific dataset?\n\nAlso, I wonder whether one could also expect variations of the other pRF parameter (pRF position), e.g. in the form of an attraction towards the fovea / screen borders / horizontal and vertical meridians. The text reports relatively high correlations between pRF center parameters at 1.5 and 3T, but this does not exclude systematic shifts in eccentricity or polar angle. The same applies to the effects of temporal filtering: can the authors exclude systematic shifts of pRF positions?\n\nI think that the figures could be more incisive if they were more compact. While I understand the importance of reporting single-participant’s data, I feel the need for gathering the data in fewer more illustrative plots. One possibility would be to use the format of scatter-plots comparing pRF parameters at 1.5 vs. 3T (so that the three subjects could be represented as different symbols within a single plot) - perhaps accompanied by a grand-average showing the expected relationship between pRF parameters and visual areas or eccentricity.\n\nIt might be worth pointing out that the bias introduced by temporal filtering is linked to the regular sequence of visual presentations. Presumably, temporal filtering would have had no effect on pRF size if the visual stimulus used for retinotopic mapping varied its position randomly - instead of sweeping across the visual field.\n\nSharing the raw data is sometimes very difficult, but clearly this is encouraged by many in the field. I wonder whether the authors could provide a justification for not providing .nii files (rather than the analyzed datasets, which are certainly sufficient for reproducing the figures but do not allow for reusing the dataset for novel investigations).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5236", "date": "26 Feb 2020", "name": "Catherine Morgan", "role": "Author Response", "response": "We’d like to thank the reviewer for their considered questions and useful suggestions on this manuscript. In our response we have reproduced the report, and commented immediately after each point. “pRF mapping provides a quantitative description of cortical representations, including the spatial representation in visual cortical areas. While pRF parameters are extensively used to quantitatively compare across subjects and conditions, we are well aware that such parameters depend on both neural factors and on nuisance variables that are related to the dynamics of BOLD. However, we lack a precise understanding of how such nuisance variables might affect each of the pRF parameters. Morgan & Schwarzkopf make an important effort in this direction and test the impact of static SNR (affected by MR field strength) and temporal SNR (affected by temporal filtering) on spatial pRF parameters in visual cortical areas. I have no major concerns with the study and only offer few suggestions that hopefully will increase the usability of the reported results: It would be useful to explicitly describe one (or more) models that predict changes of pRF parameters with SNR. I feel one intuitive (and probably wrong) model invokes a non-linear thresholding effect. The logic is, approximately, as follows: when BOLD responses are stronger, each voxel responds to a larger range of stimulus position and the pRF size is consequently increased. Is this one of the hypotheses that the study intends to test (and falsify)? This particular model would predict that beta values (indexing BOLD response amplitude) and pRF size estimates correlate positively. Since both indices were collected here, could the authors measure this (lack of) correlation?”>>Thank you for this important question. A stronger response amplitude could potentially appear as a larger pRF because visual field locations farther from the pRF centre respond to the stimulus. However, this is unlikely to manifest as a positive correlation with the response amplitude (beta) parameter because the pRF model is scale-invariant, that is, the pRF parameters are estimated by correlation between predicted and observed time series and thus theoretically only the pRF shape matters to estimation.In fact, the opposite relationship, that pRF size estimates are larger with low beta parameters, is actually more realistic: response amplitudes are linked to signal-to-noise and thus goodness-of-fit. Under low noise conditions, a small pRF would show a clean response time series with very clearly defined peaks. In a noisier pRF, this time series would however be contaminated and so the peaks of the response would be less clearly defined. In order to fit a pRF model, the algorithm will therefore produce an artifactual bias on pRF size estimates. We have already seen this relationship in previous work. However, for the sake of completeness we now conducted the suggested correlation analysis between beta parameters and pRF size at the single-vertex level. This confirms that the second model is indeed correct: across participants and visual regions of interest there is a negative correlation between pRF size estimates and beta parameters (Spearman’s rho ranging between -0.17 and -0.5 for most cases, with the exception of V3A where the small amount of data may skew the results). In other words, when response amplitude is low, pRFs tend to be estimated as larger. Unfortunately, this analysis is complicated by several factors: the data are highly heteroscedastic (there is far more variability in response amplitude for small pRFs than large ones), a small but considerable proportion of beta parameters is negative (we believe these are related to artifactual pRF estimates with negative BOLD responses due to large blood vessels or edge-of-stimulus artifacts), and there is also a strong but incomplete ceiling artifact as many pRF sizes cluster at a small size. Nevertheless, we believe due to its consistency and robustness to thresholding this relationship probably correct. We now briefly discuss this issue in the introduction of the revised manuscript. Please note that because we find no consistent difference in pRF size between scanner sites, there is no evidence that this link has any bearing on the conclusions of our present study. Therefore, and because of their complexity, we chose not to present and discuss the results of this control analysis in the revised manuscript. However, the analysis script for this is now included in our online repository. “In addition, this model would incorrectly predict larger pRF size estimates at higher MR fields, under the assumption that response amplitude is positively related to SNR. Could the authors report whether this assumption (larger beta-values in the higher SNR, i.e. 3T, dataset) is correct for their specific dataset?” >> As the reviewer correctly points out, in MRI, the signal-to-noise ratio (SNR) is directly proportional to main magnetic field strength (and voxel volume) (Edelstein, Glover, Hardy, & Redington, 1986). Therefore power to detect BOLD activation theoretically increases with static magnetic field (or larger voxel sizes), or where this is not possible/desirable, longer scan times. However, noise in fMRI data has multiple contributions (physiological, thermal and system related) and therefore the relationship between the temporal signal to noise ratio (TSNR) in a fMRI time course has a non-linear relationship with static SNR. This has been demonstrated theoretically and empirically (Murphy, Bodurka, & Bandettini, 2007) showing that any gains in static SNR, from e.g. increased field strength, will not translate to proportional gains in TSNR due to a limit where physiological noise dominates. Since response amplitude (beta) is related to goodness-of-fit, we assume that betas are indeed greater at 3T. We now conducted the suggested analysis and this confirms that prediction: betas are larger at 3T at least in the early visual areas showing a similar pattern as goodness-of-fit. We added this figure to what is now Supplementary Figure S1. But importantly, as we explained above, larger betas do not result in larger pRF size estimates but rather the opposite. Despite this, we found no consistent differences in pRF size estimates between the two scanner sites. “Also, I wonder whether one could also expect variations of the other pRF parameter (pRF position), e.g. in the form of an attraction towards the fovea / screen borders / horizontal and vertical meridians. The text reports relatively high correlations between pRF center parameters at 1.5 and 3T, but this does not exclude systematic shifts in eccentricity or polar angle. The same applies to the effects of temporal filtering: can the authors exclude systematic shifts of pRF positions? “ We would not predict any systematic relationship between pRF position and magnetic field strength but this is an interesting suggestion. There does not appear to be any strong evidence of such a systematic bias in the maps at least (please refer to the example maps we included – an interested reader may be able to reproduce them using our shared analysis scripts). The biggest differences in eccentricity maps between the two sites appear to be additional voxels within the foveal region in the 3T data, rather than an obvious shift in the map. We however understand that this visual inspection of the maps is only a very coarse analysis. A quantitative analysis would be to plot visual field positions directly against one another. For reasons that are outside the scope of this discussion, it is imperative that this comparison must be done using an independent criterion for determining which data points we compare between the two sites. We therefore determined the cortical distance of each vertex in the spherical surface model from a point chosen near the occipital pole as a proxy of ground truth eccentricity. We then quantified the mean eccentricity within 5 mm cortical distance bins for the two sites. This revealed no obvious systematic differences in eccentricity between the two sites. We believe this analysis is too complex to be included in the main publication but we included the code for it in our public repository and made this figure publicly available: https://osf.io/k4dcr/ “I think that the figures could be more incisive if they were more compact. While I understand the importance of reporting single-participant’s data, I feel the need for gathering the data in fewer more illustrative plots. One possibility would be to use the format of scatter-plots comparing pRF parameters at 1.5 vs. 3T (so that the three subjects could be represented as different symbols within a single plot) - perhaps accompanied by a grand-average showing the expected relationship between pRF parameters and visual areas or eccentricity.” >> Thank you for this observation. We agree both you and the other reviewer make a good point and we have now consolidated the individual plots into group average plots, and moved the individual figures into the supplementary material. We also like your idea of scatter plots but we believe they would also be somewhat complex as we would ideally also need to denote the different eccentricities (perhaps with a colour code). Especially for measures that do not change linearly with eccentricity (like beta or goodness-of-fit) this would make for very dense plots. It might be worth pointing out that the bias introduced by temporal filtering is linked to the regular sequence of visual presentations. Presumably, temporal filtering would have had no effect on pRF size if the visual stimulus used for retinotopic mapping varied its position randomly - instead of sweeping across the visual field. >> This is a very good suggestion and we now discuss this in the revised manuscript. However, please also keep in mind that other work by ourselves (1, currently under peer review, but a preprint is available here: https://doi.org/10.1101/821918) and of course also your own work 2has shown that pRF size estimates differ between random and ordered designs. This issue was also discussed in a study on tonotopic pRF mapping3. It is possible that temporal filtering would not show the effect we report here for a random design but there are probably additional factors linking pRF size with randomisation. For instance, we showed in Infanti & Schwarzkopf that whether or not pRF size is over- or underestimated in ordered relative to random designs also depends on the stimulus parameters. Sharing the raw data is sometimes very difficult, but clearly this is encouraged by many in the field. I wonder whether the authors could provide a justification for not providing .nii files (rather than the analyzed datasets, which are certainly sufficient for reproducing the figures but do not allow for reusing the dataset for novel investigations). >> please refer to our response to reviewer 1 to answer this point. In short, there are both legal and ethical barriers to our sharing of raw data files. However, we want to stress that all of the time series information necessary for reproducing the pRF analysis, reanalysing it with a different software, or conducting additional novel analyses is available in our public repository. For anyone seeking to conduct analysis that are not possible with the shared data we are willing to make these data available upon request provided the participants consent to this use. Response References 1. Infanti, E. & Schwarzkopf, D. S. Mapping sequences can bias population receptive field estimates. bioRxiv 821918 (2019) doi:10.1101/821918.2. Binda, P., Thomas, J. M., Boynton, G. M. & Fine, I. Minimizing biases in estimating the reorganization of human visual areas with BOLD retinotopic mapping. J. Vis. 13, 13–13 (2013).3. Thomas, J. M. et al. Population receptive field estimates of human auditory cortex. NeuroImage 105, 428–439 (2015)." } ] } ]	1	https://f1000research.com/articles/8-1681
https://f1000research.com/articles/9-136/v1	24 Feb 20	{ "type": "Opinion Article", "title": "BioHackathon 2015: Semantics of data for life sciences and reproducible research", "authors": [ "Rutger A. Vos", "Toshiaki Katayama", "Hiroyuki Mishima", "Shin Kawano", "Shuichi Kawashima", "Jin-Dong Kim", "Yuki Moriya", "Toshiaki Tokimatsu", "Atsuko Yamaguchi", "Yasunori Yamamoto", "Hongyan Wu", "Peter Amstutz", "Erick Antezana", "Nobuyuki P. Aoki", "Kazuharu Arakawa", "Jerven T. Bolleman", "Evan E. Bolton", "Raoul J. P. Bonnal", "Hidemasa Bono", "Kees Burger", "Hirokazu Chiba", "Kevin B. Cohen", "Eric W. Deutsch", "Jesualdo T. Fernández-Breis", "Gang Fu", "Takatomo Fujisawa", "Atsushi Fukushima", "Alexander García", "Naohisa Goto", "Tudor Groza", "Colin Hercus", "Robert Hoehndorf", "Kotone Itaya", "Nick Juty", "Takeshi Kawashima", "Jee-Hyub Kim", "Akira R. Kinjo", "Masaaki Kotera", "Kouji Kozaki", "Sadahiro Kumagai", "Tatsuya Kushida", "Thomas Lütteke", "Masaaki Matsubara", "Joe Miyamoto", "Attayeb Mohsen", "Hiroshi Mori", "Yuki Naito", "Takeru Nakazato", "Jeremy Nguyen-Xuan", "Kozo Nishida", "Naoki Nishida", "Hiroyo Nishide", "Soichi Ogishima", "Tazro Ohta", "Shujiro Okuda", "Benedict Paten", "Jean-Luc Perret", "Philip Prathipati", "Pjotr Prins", "Núria Queralt-Rosinach", "Daisuke Shinmachi", "Shinya Suzuki", "Tsuyosi Tabata", "Terue Takatsuki", "Kieron Taylor", "Mark Thompson", "Ikuo Uchiyama", "Bruno Vieira", "Chih-Hsuan Wei", "Mark Wilkinson", "Issaku Yamada", "Ryota Yamanaka", "Kazutoshi Yoshitake", "Akiyasu C. Yoshizawa", "Michel Dumontier", "Kenjiro Kosaki", "Toshihisa Takagi", "Rutger A. Vos", "Hiroyuki Mishima", "Shin Kawano", "Shuichi Kawashima", "Jin-Dong Kim", "Yuki Moriya", "Toshiaki Tokimatsu", "Atsuko Yamaguchi", "Yasunori Yamamoto", "Hongyan Wu", "Peter Amstutz", "Erick Antezana", "Nobuyuki P. Aoki", "Kazuharu Arakawa", "Jerven T. Bolleman", "Evan E. Bolton", "Raoul J. P. Bonnal", "Hidemasa Bono", "Kees Burger", "Hirokazu Chiba", "Kevin B. Cohen", "Eric W. Deutsch", "Jesualdo T. Fernández-Breis", "Gang Fu", "Takatomo Fujisawa", "Atsushi Fukushima", "Alexander García", "Naohisa Goto", "Tudor Groza", "Colin Hercus", "Robert Hoehndorf", "Kotone Itaya", "Nick Juty", "Takeshi Kawashima", "Jee-Hyub Kim", "Akira R. Kinjo", "Masaaki Kotera", "Kouji Kozaki", "Sadahiro Kumagai", "Tatsuya Kushida", "Thomas Lütteke", "Masaaki Matsubara", "Joe Miyamoto", "Attayeb Mohsen", "Hiroshi Mori", "Yuki Naito", "Takeru Nakazato", "Jeremy Nguyen-Xuan", "Kozo Nishida", "Naoki Nishida", "Hiroyo Nishide", "Soichi Ogishima", "Tazro Ohta", "Shujiro Okuda", "Benedict Paten", "Jean-Luc Perret", "Philip Prathipati", "Pjotr Prins", "Núria Queralt-Rosinach", "Daisuke Shinmachi", "Shinya Suzuki", "Tsuyosi Tabata", "Terue Takatsuki", "Kieron Taylor", "Mark Thompson", "Ikuo Uchiyama", "Bruno Vieira", "Chih-Hsuan Wei", "Mark Wilkinson", "Issaku Yamada", "Ryota Yamanaka", "Kazutoshi Yoshitake", "Akiyasu C. Yoshizawa", "Michel Dumontier", "Kenjiro Kosaki", "Toshihisa Takagi" ], "abstract": "We report on the activities of the 2015 edition of the BioHackathon, an annual event that brings together researchers and developers from around the world to develop tools and technologies that promote the reusability of biological data. We discuss issues surrounding the representation, publication, integration, mining and reuse of biological data and metadata across a wide range of biomedical data types of relevance for the life sciences, including chemistry, genotypes and phenotypes, orthology and phylogeny, proteomics, genomics, glycomics, and metabolomics. We describe our progress to address ongoing challenges to the reusability and reproducibility of research results, and identify outstanding issues that continue to impede the progress of bioinformatics research. We share our perspective on the state of the art, continued challenges, and goals for future research and development for the life sciences Semantic Web.", "keywords": [ "BioHackathon", "Bioinformatics", "Semantic Web", "Web Services", "Ontology", "Visualization", "Databases", "Linked Open Data", "Metadata", "Workflows" ], "content": "Abbreviations\n\nAPI, Application Programming Interface; BH15, BioHackathon 2015; CUI, Concept Unique Identifier; CV, Controlled Vocabulary; DOID, DO IDentifier; DPA, Disease-Phenotype Association; EHR, Electronic Health Records; FAIR, Findable, Accessible, Interoperable and Reusable; GDA, Gene-Disease Association; GPM, General Process Model; LIMS, Laboratory Information Management System; MSEA, Metabolite Set Enrichment Analysis; ORCID, Open Researcher and Contributor ID; NLP, Natural Language Processing; NMR, Nuclear Magnetic Resonance; VG, genomic Variation Graph.\n\nBAO, BioAssay Ontology; CDAO, Comparative Data Analysis Ontology; ChEBI, Chemical Entities of Biological Interest; CHEMINF, CHEMical INFormation ontology; DC, DCT, Dublin Core, Dublin Core Terms; DO, Disease Ontology; EFO, Experimental Factor Ontology; EpSO, Epilepsy and Seizure Ontology; ERO, Eagle-i Resource Ontology; EXACT, Experiment ACTions ontology; EXPO, Ontology of scientific experiments; FMA, Foundational Model of Anatomy; FOAF, Friend Of A Friend; GO, Gene Ontology; HPO, Human Phenotype Ontology; IAO, Information Artifact Ontology; LABORS, LABoratory Ontology for Robot Scientists; MOD, Metadata for Ontology Description; MP, Mammalian Phenotype ontology; OA, Open Annotation ontology; OBAN, Ontology of Biomedical AssociatioN; OBI, Ontology for Biomedical Investigations; OMV, Ontology Metadata Vocabulary; ORDO, Orphanet Rare Disease Ontology; ORTH, ORTHology ontology; PATO, Phenotypic quality ontology; PICO, Patient Intervention Comparison Outcome; PIERO, Partial Information of chemical transformation; RO, Relations Ontology; SIO, Semanticscience Integrated Ontology; SIRO, Sample, Instrument, Reagent, Objective; SMART Protocols, SeMAntic RepresenTation for experimental protocols; UMLS, Unified Medical Language System.\n\nBTMG, Biomedical Text Mining Group at the NIH; DBCLS, Database Center for Life Science; EBI, European Bioinformatics Institute; GA4GH, Global Alliance for Genomics and Health; HGNC, HUGO Gene Nomenclature Committee; jPOST, Japan Proteome Standard Repository/Database; LOV, Linked Open Vocabularies; NBDC, National Bioscience Database Center; NCBI, National Center for Biotechnology Information; NCBO, National Center for Biomedical Ontology; NESCent, National Evolutionary Synthesis Center; NIH, National Institutes of Health; OBO Foundry, Open Biomedical Ontologies Foundry; Open PHACTS, Open Pharmacological Concept Triple Store; PDBj, Protein Database Japan; RDA, Research Data Alliance.\n\nCWL, Common Workflow Language; DisGeNET, Disease Gene Network; GEO, Gene Expression Omnibus; HUPO-PSI, Human Proteome Organization Proteomics Standards Initiative; KEGG-OC, Kyoto Encyclopedia of Genes and Genomes – Orthologous Clusters; LSDB Archive, Life Science Database Archive; MBGD, Microbial Genome Database; MeKO, Metabolite profiling database for Knock-Out mutants in Arabidopsis; OLS, Ontology Lookup Service; OMA, Orthologous MAtrix; OMIM, Online Mendelian Inheritance in Man; ORKA, Open, Reusable Knowledge graph Annotator; PASSEL, Peptide AtlaS SRM Experiment Library; PMR, Plant and Microbial Metabolomics Resource; PRIDE, PRoteomics IDEntifications database ; QfO, Quest for Orthologs; SADI, Semantic Automated Discover and Integration; SIDER, SIDe Effect Resource; SWIT, Semantic Web Integration Tool.\n\nBED, Browser Extensible Data; HPC, High Performance Computing; HTTP, HyperText Transfer Protocol; JSON, JavaScript Object Notation; JSON-LD, JSON – Linked Data; LOD, Linked Open Data; OWL, Web Ontology Language; RDF, Resource Description Framework; RDFa, RDF in Attributes; RML, RDF Modeling Language; SAM/BAM, Sequence Alignment/Map, Binary Alignment/Map; SHA, Secure Hash Algorithm; SPARQL, SPARQL Protocol and RDF Query Language; TPF, Triple Pattern Fragments1; URI, Universal Resource Identifier; VCF, Variant Call Format; YAML, YAML Ain’t Markup Language; XML, eXtensible Markup Language.\n\n\nBackground\n\nThe past few years have yielded considerable progress in the development and application of fundamental digital technologies that support research in the life sciences2, including ontologies and Linked Open Data (LOD), semantic web services, natural language processing, and tooling for workflows and virtualization. While these technologies are useful for life sciences research, key to their long-term success lies in community agreements that foster standardization and interoperability2. In an effort to coordinate the social and technological aspects of in silico life sciences research, the authors convened at the 2015 edition of the BioHackathon (BH15), an event that aims to create a highly collaborative environment to explore, evaluate, and implement solutions to the problems of data publication, integration, and reuse3–6. A hackathon is a type of software development lifecycle model featuring problem-focused development via intensive, time-limited, self-organized group activities, typically involving programmers and various types of collaborators7. The hackathon methodology has been shown to be productive in a variety of biomedical fields, including rehabilitative healthcare8, biological data science9, neuroscience10, computer-aided differential diagnosis11, stroke detection12, standards specification in systems and synthetic biology13, data science for knowledge discovery in medicine14, medical device innovation15, enrichment of biodiversity data16, and teaching genomics17. BH15 was held in Nagasaki, Japan, over the period of September 14th to 18th 2015, and was hosted by the National Bioscience Database Center (NBDC,18) and the Database Center for Life Science (DBCLS,19) to promote interoperability of life sciences databases in Japan. Researchers and developers from around the world participated by invitation. BH15 was preceded by a public symposium featuring new research and updates from the participants. BH15 involved 80 individuals from 12 countries and a wide variety of backgrounds, including computer programmers, bioinformaticians, biocurators, ontologists, biological scientists, systems biologists, data scientists, and linguists.\n\nHere, we present selected outcomes from BH15, self-organized by the participants in projects around different topics, which we discuss in the following sections. At the highest level, the contours of these topics are, broadly, i) life sciences data, including genotypes and phenotypes, orthology and phylogeny, proteomics, metabolomics, and biochemical molecular; and ii) research methods, i.e. the technologies that support in silico analysis in the life sciences, including data retrieval and querying, natural language processing, reproducibility, and semantic metadata. Under these broad topics, we identify various themes within which specific activities took place. These topics and themes are illustrated in Figure 1. The activities and their scopes were identified by the participants through self-organization following Open Space technology20. As such, the commitment of the participants to any particular activity was somewhat free-wheeling, and so we report the outcomes collectively, rather than subdivided by participant teams. The results of the work reported here are relevant both to evaluate the current state of the relevant technologies and problem areas in the life sciences, and to help the field understand the potential and problems of future research and development efforts.\n\n\nLife sciences data\n\nVariation graph construction. In the context of the Global Alliance for Genomics and Health (GA4GH,21) there is a challenge to build genomic variation graphs (VG). A genomic variation graph represents all “common” genetic variation, providing a means to stably name and canonically identify each variant. At BH15, we modeled such graphs using RDF semantics. Taking the 1000 Genomes project phase 3 Variant Call Format (VCF) files and the GRCh37 human reference genome we built a variant graph using the VG tool22. Such a VG graph corresponds to just fewer than 2 billion triples. It was loaded inside 67 minutes on a server from 2013 that had 64 AMD X86_64 cores, 256 GB ram, and 3 TB of consumer-quality SSD storage without specific tuning. The SPARQL database disk footprint with indexes was 49 GB, i.e. double the disk space consumed by the raw VG tool files. This shows that a modern SPARQL database does not require exorbitant resources to be able to index and load a variant graph of interest to the medical community. We also demonstrated that a number of queries on the graph executed within reasonable times. This work was contributed to the VG development team and incorporated into the VG release 1.4.0. At BH15, the standard API developed by the core API team of the GA4GH was implemented as a service running on top of a SPARQL endpoint.\n\nVariant call transformation. VCF is a standard for text files that store gene sequence variations and is used for large-scale genotyping and DNA sequencing projects. Converting a single high-throughput sequence dataset, e.g. a VCF file but more so a very large database such as the Ensembl Variation Database, into RDF results in a number of triples that may be unmanageable for a small bioinformatics lab, even if backed by current hardware. However, data can also be considered in a more dynamic way, if we abstract the concept of data generation to, for instance, some bioinformatics analysis or pipeline, where new data can be generated on the fly as a result of some computation over existing information or files. To this end we prototyped a real-time system to transform VCF into RDF and query it by SPARQL. With JRuby we could use the original samtools/htsjdk libraries for manipulating VCF, BED, SAM/BAM files. With this approach, we could quickly prototype our solution and defer the development of proper Java libraries sharable by alternative approaches and/or applications. Our approach allows generating virtual endpoints over multiple VCF files, combining the simplicity of native file formats with the power of the SPARQL language, significantly improving the way we link and query heterogeneous information. An implementation of such a system was conceived during the 1st RDF summit in 2014 at the DBCLS in Tokyo and further developed during BH15. The system23 was based on de facto standard frameworks, such as Ruby RDF24 and OpenSesame25, which facilitate the generation and transformation of RDF based data and the processing of SPARQL algebra and queries.\n\nPhenotype ontology translation. Precision medicine aims to provide patient-tailored diagnostics, prognostics, treatments, and prevention. Part of the strategy to precision medicine involves more precise clinical phenotyping. The Human Phenotype Ontology (HPO) is an ontology of abnormal phenotypes associated with human disease26. Originally aimed to describe Mendelian genetic diseases, it has since been expanded to cover phenotypes associated with rare and common diseases. The availability of phenotype terms expressed in the Japanese language is key to its application in text mining Electronic Health Records (EHR) in Japan.\n\nThe development project of HPO-Japanese was initiated prior to BH15 in cooperation with the HPO teams (Dr. Peter Robinson, Dr. Melissa Haendel, Dr. Nicole Vasilevsky, and Dr. Tudor Groza). We translated English terms into Japanese by exact matches to existing English-Japanese dictionaries, including the Elements of Morphology – Standard Terminology (Japanese ed.), the Japanese Association of Medical Sciences Medical Term Dictionary, the Life Science Dictionary (LSD), and general dictionaries. The total number of terms translated is 11,425. Elements of Morphology – Standard Terminology (Japanese ed.) covers ~400 terms (3.5%), the Japanese Association of Medical Sciences Medical Term Dictionary covers 1,807 terms (15.8%). The remaining terms need to be curated by experts. We are now compiling several translated terms as curated HPO-Japanese. Once completed, HPO-Japanese will be open and available so that precise and standardized phenotyping can be undertaken using Japanese EHR text and which can be directly linked to the international resources and research systems through HPO identifiers.\n\nOrthology ontology development and application. Orthologs are defined as genes derived from a common ancestral gene by speciation. Orthology information can play a central role in predicting gene function in newly sequenced genomes and can also help unravel the evolutionary history of genes and organisms. Orthology resources have been represented in a variety of formats, including the OrthoXML27 that is used by several orthology databases such as InParanoid28, Orthologous MAtrix (OMA,29), and TreeFam30. The interest in exchanging orthology data with other communities has provided the impetus for research on applying the Semantic Web and using common ontologies for making the meaning of the content explicit. Thus, on the basis of previous studies on the semantic representation of orthology31,32, we made efforts during BH15 towards semantic standardization of orthology content33.\n\nWe developed the Orthology Ontology (ORTH34, and 35) to capture essential concepts pertaining to orthology, including clusters of orthologs derived from speciation events. ORTH was designed following best practices in ontology engineering, i.e., reusing related ontologies such as the Semanticscience Integrated Ontology (SIO,36), the Relations Ontology (RO,37), and the Comparative Data Analysis Ontology (CDAO,38). We used the Semantic Web Integration Tool (SWIT,39 and40), a generic tool for generating semantic repositories from relational databases and XML sources, to convert InParanoid, OMA, and TreeFam datasets in OrthoXML format into RDF. More details and sample queries for the datasets using ORTH are on the source code repository41,42.\n\nAlthough the standard mapping and transformation by SWIT was largely able to transform the content of the three databases, though a few resource-specific rules were necessary because: (1) OrthoXML offers generic tags that are used by orthology databases in a heterogeneous way, e.g. for describing the taxonomic range of a cluster of orthologs; and (2) different orthology resources use identifiers of genes or proteins from different databases, so the corresponding prefixes for URIs had to be adapted. The next steps include: (1) evaluation of the results by the Quest for Orthologs (QfO,43) community, which could lead to the development of a QfO semantic infrastructure for sharing orthology resources; (2) examining the interoperability of semantic orthology datasets using additional databases such as UniProt44, KEGG OC45, and the Microbial Genome Database (MBGD,46); and (3) developing applications and tools for comparative analysis of genomes/proteomes utilizing the ORTH.\n\nMolecular evolutionary process calibration. Not only qualitative but also quantitative representation of evolutionary events, i.e. on a time axis, among organisms is important for evolutionary biology. However, the adoption of Semantic Web technologies is lagging behind in domains of the biological sciences outside of the conventional scope of BH15. For example, in recent years several hackathons and other meetings have been held to address challenges of data mobilization47 and integration in phyloinformatics48,49 and biodiversity informatics16 that uncovered a paucity of web services that deliver ontologized, or even machine-readable, data on fossil specimens. Although expected waiting times between speciation events can be modeled50, fossils are needed for calibrating phylogenies to absolute time axes49,51,52, e.g. to detect nucleotide substitution rate shifts coinciding with evolutionary events such as speciations, which generate orthology, and gene duplications, which generate paralogy.\n\nRecently, a working group at the National Evolutionary Synthesis Center (NESCent,53) initiated a project to address this54 and to establish a database of reference fossils with a web service API55. To evaluate whether this new resource can indeed be usefully applied in the analysis of molecular data we developed a proof-of-concept pipeline55 (based on Bio::Phylo56 and SUPERSMART57) that includes a reconciliation between fossil taxa from the FossilCalibrations database and extant taxa from in the TreeFam orthology database. The steps are as follows:\n\n1. Download a data dump release from TreeFam.\n\n2. For each TreeFam gene family, fetch fossils from FossilCalibrations through the API. This was done by querying for the taxonomic names, e.g. “Mammalia”, that are applied to internal node labels in gene family trees.\n\n3. Apply the fossil ages as calibration points for a penalized likelihood analysis using r8s58.\n\n4. Using the produced ‘ratogram’ (a phylogenetic tree whose branch lengths are proportional to inferred substitution rates, one of the results produced by the r8s analysis), calculate the substitution rate as a function of time since the most recent gene duplication event.\n\nThe rationale for this pipeline was that the general model of gene duplication followed by neo- or subfunctionalization59 suggests that reconstructed substitution rates (which are retrospective, and based on accumulated fixed mutations) should be elevated in novel gene copies that are either under relaxed or under directional selective pressure. Hence, we would expect to see elevated substitution rates following a duplication event, which should taper off over time. Given that baseline substitution rates differ between lineages we performed an assessment of whether this prediction could be detected confined to a single lineage, that of C. elegans. Figure 2 suggests that this is indeed the case (this is in essence a different way of obtaining, roughly, some of the findings of60). As a proof of concept to test whether it is possible to include fossil data from this new resource we conclude that this is indeed possible, but we note several drawbacks:\n\nThe FossilCalibrations database makes its data available as simple JSON. This is convenient for programmers but it also means that certain concepts used in the JSON are ambiguous as they are not linked to any controlled vocabulary or ontology.\n\nThe distinction between stem and crown fossils is made using magic numbers whose values and their meanings are poorly documented (we could only discover their semantics by inspecting the source code of FossilCalibrations).\n\nSome of the taxon names used by FossilCalibrations are not scientific names from any explicitly identified taxonomy. For example, some fossil calibration points have names such as “Chimpanzee-Human”, or “Humanity”. Such names are difficult to resolve using taxonomic name resolution services.\n\nThere are large biases in taxon sampling in the database. In fossil databases this is nearly inevitable as some taxa fossilize much better than others, but even where a relatively rich fossil record is known to exist, e.g. in the sea urchins (Takeshi Kawashima, pers. comm.), no records were available in the database.\n\nThe first three drawbacks we identified can all be traced back to poorly defined semantics, which we therefore characterize as the key current issue in LOD representation of fossil specimens. To fill this gap, firstly we need to semantically curate FossilCalibrations data manually, which of course may take time, then export curated information in RDF so that analyses proposed in this section can be integrated in the automated pipeline.\n\nProtein semantic representation. Many datasets on the Semantic Web are available as RDF, but often lack the explicit model-theoretic semantics provided by languages such as OWL. For complex datasets, the additional semantics of OWL, which includes assertions of disjointness, i.e. the explicit semantic distinction between classes and their instances, and axioms restricting the use of classes and object properties, may be particularly beneficial. The main limitation of languages such as OWL is that querying them is often highly computationally intensive and therefore not feasible for large datasets. Our aim was to evaluate how well formal languages like OWL scale in representing very large datasets. We chose the UniProt database44, as it currently constitutes one of the larger RDF datasets, is used throughout biology, and has rigorous quality checks. Our aim was to find a representation of proteins and their functions using OWL. As automated reasoning over OWL knowledge bases is highly complex (2-NEXPTIME complete), we limited ourselves to the OWL 2 EL profile. However, widely used ontology design patterns for representing functions are not expressible in OWL 2 EL, as certain types of restrictions (in particular universal quantification) do not fall within the OWL 2 EL expressivity. As a consequence of these limitations, we decided to develop a novel representation pattern for asserting that proteins have a function that would fall in OWL 2 EL and would enable us to convert all of UniProtKB into OWL (though for testing purposes we converted only a subset on the order of 105 OWL axioms). Specifically, given proteins XYZ, we generate the following classes:\n\nClass XYZ (instances of this class are individual proteins)\n\nClass XYZ_all (instances are the sets of all XYZ proteins in the universe; intuitively, only one instance of this class can ever exist)\n\nClass XYZ_isoform for all isoforms of XYZ\n\nClass XYZ_generic (the 'generic' form of the protein, i.e., a group of orthologous proteins)\n\nWe also generate the following axioms (here expressed in Manchester OWL Syntax):\n\nXYZ SubClassOf: XYZ_generic\n\nXYZ_isoform SubClassOf: XYZ\n\nXYZ_isoform SubClassOf: isoform-of some XYZ\n\nXYZ SubClassOf: member-of some XYZ_all\n\nXYZ_all SubClassOf: { xyz } i.e., XYZ_all is a singleton class, and lower-case xyz is a new constant symbol that is newly introduced for each axiom of that type\n\nXYZ_all SubClassOf: has-member only XYZ (XYZ_all is homogenic)\n\nOf these axioms, only the last axiom (XYZ_all is homogenic) is not expressible in OWL 2 EL, while all other axioms can be expressed in the OWL 2 EL profile. We have converted several types of proteins from UniProtKB using this approach and evaluated queries and query time. However, a thorough analysis on how well this approach scales to ontologies of the size of UniProtKB is left for future work. The source code developed for this project is available at our source code repository61,62.\n\nProteome assay annotation. In proteomics, expressed proteins are usually identified by mass spectrometry. In most common workflows, proteins are digested into peptides with a protease. The peptides are ionized and then fragmented. Their precursor mass-to-charge ratios and fragment ion spectra are experimentally measured and compared with theoretical masses and fragmentation patterns of peptides calculated from a protein database. Information about experimental protocols and data analysis methods is thus important for understanding the raw and processed data. An identified protein list has substantial amounts of metadata such as labels used for quantification, e.g. iTRAQ,63, or SILAC,64, protease used for protein digestion (most commonly trypsin), pre-separation method (LC, 2D-gel electrophoresis, etc.), ionization and ion detection method of the mass spectrometer (MALDI-TOF-TOF, etc.), peak-processing software (ProteoWizard,65; MaxQuant,66; etc.), protein database used for theoretical peptide mass calculation (UniProt,44; Ensembl,67; etc.), database search software for peptide-spectral matches (Mascot,68; X!Tandem,69; MaxQuant,66; etc.), and parameters and thresholds of the software. These experimental protocol- and data analysis method-related terms are necessary metadata for submissions to proteome databases/repositories.\n\nTo describe these metadata, the Human Proteome Organization Proteomics Standards Initiative (HUPO-PSI) has developed the PSI-MS controlled vocabulary70 and ProteomeXchange71, which is a consortium of mass spectrometry proteomics data repositories including PRIDE, the Peptide Atlas SRM Experiment Library (PASSEL,72), and MassIVE73, has established a core set of metadata for dataset deposition using PSI-MS.\n\nThe Japanese proteome community is now developing the Japan Proteome Standard (jPOST) repository74, which is a mass spectrometry proteomics data repository. The salient feature of jPOST is the ability to re-analyze data from deposited raw data; by using raw data and a jPOST-original re-analysis workflow, the community plans to integrate data from various experiments to construct a standardized proteome database (jPOST database). Original analytical results from submitters are not suitable for integration because they were performed using various different protein databases and peak-identification/database search software with various different parameters.\n\nFor re-analysis, it is necessary to describe detailed information about experimental procedures. However, current controlled vocabularies (CVs) such as PSI-MS are insufficient for metadata description, and so we have attempted to reorganize and extend the current CVs for jPOST. At BH15, we enumerated required categories of metadata, such as Instrument mode and Quantification platform, and collected vocabularies with the cooperation of experimental proteomics scientists. The collected vocabularies were mapped to existing CVs where possible, and we began to develop an ontology for unmapped vocabularies75. We also developed an RDF schema based on the CVs and ontology (Figure 3) for jPOST datasets. Constructing an ontology that is compatible with existing CVs such as PSI-MS is important for integrating jPOST data with other proteomics data stored in the databases of the ProteomeXchange Consortium71. In addition, by using common ontologies/CVs such as Taxonomy and disease name and a standardized data model like RDF, the proteomics datasets can also be linked and integrated with datasets derived from other technologies such as transcriptomics and epigenomics.\n\nTools for metabolite identification and interpretation. Metabolomics is the biochemical analysis of all low-molecular-weight metabolites in a biological system, i.e. the metabolome. Owing to the chemical diversity and complexity of the metabolome, no single analytical platform can detect all metabolites in a sample simultaneously. Current state-of-the-art approaches for measuring metabolites and maximizing metabolite coverage require integration of multiple analytical platforms, data pre-processing methods, effective metabolite annotation, and data interpretation76,77. Given that the most commonly used analytical and data pre-processing methods have been comprehensively reviewed78–80, we will not discuss them here, but rather focus on downstream analyses such as pathway analysis, and effective data interpretation.\n\nScientists in natural products chemistry use the accurate mass and chemical shifts in Nuclear Magnetic Resonance (NMR) spectra to elucidate the structure of unknown natural chemical compounds. In contrast, researchers in metabolomics commonly try to provisionally identify chromatographic peaks by comparing their retention time (or retention indices), and/or the mass spectra, with those present in a mass spectral library database generated from the data of authentic standards81. The Metabolomics Standards Initiative defined four levels of reporting metabolite identification and annotation: identified metabolite (Level 1), putatively annotated metabolites (Level 2), putatively annotated metabolite classes (Level 3), and unknown compounds (Level 4)82. This indicates that the confidence levels of metabolite identification reported in metabolomics studies can vary largely, because of different extraction protocols, different instruments and measurement parameters, different pre-processing methods, and the diversity of annotation expertise83. This hampers the reusability/reanalysis of published metabolomics datasets, although there are public repositories for metabolomics data such as MetaboLights84 and MetabolomeExpress.org85.\n\nBiological interpretation of changes in metabolite levels is very important and is still challenging, because such metabolite pools are the resulting output of many biological processes. To facilitate biological interpretation by existing biological knowledge, e.g. biochemical pathways, pathway-based analysis like Metabolite Set Enrichment Analysis (MSEA,86) is available. This approach highly depends on predefined biological pathways such as KEGG87, Pathway Commons88, BioCyc89, and WikiPathways90. Molecular interactions can be regarded as a network by calculating association between molecules in omics data. Correlation-based approaches are behind for construction of association networks such as gene co-expression networks in transcriptomics (for example, see 91,92).\n\nThere are many software tools for pathway visualization and integration of different omics data (for example, see 93). Examples include KEGG Mapper94, KEGGViewer95, PathVisio96, WikiPathways App97, and KEGGScape98. Metscape is a Cytoscape App for network analysis and visualization of gene-metabolite associations99. MetaMapR100 can be used for integrating biochemical reaction with chemical structural and mass spectral similarity to analyze pathway-independent associations including unknown metabolites. MetaboAnalyst101 provides a user-friendly, web-based analytical platform for metabolome data pre-processing, normalization, statistical analysis, and metabolite annotation. DeviumWeb102 is also a user-friendly web application for integrating statistical multivariate analysis with biochemical domain knowledge using R-Shiny103, a web application framework for R.\n\nPlant metabolome database development. Unlike compound and mass spectral databases such as KEGG87 and MassBank104, metabolite-profile oriented databases still remain relatively undeveloped and under-used in plants81. The data and metadata for more than 140 mutants of Arabidopsis thaliana, an important model plant, are archived at the Plant and Microbial Metabolomics Resource (PMR,105). It is a flexible database that is designed for data sharing in metabolomics and implements data analysis tools106. Information on phenotypic screening of Arabidopsis chloroplast mutants using assays of amino acids and fatty acids of more than 10,000 T-DNA insertion mutants using mass spectrometry are stored in Chloroplast 2010107–109.\n\nWe recently developed a new database, the Metabolite profiling database for Knock-Out mutants in Arabidopsis (MeKO,110), to facilitate improvement of gene annotation. The MeKO database111 can be used to browse and visualize metabolomic data, containing images of mutants, data on differences in metabolite levels, and the results of statistical data analyses. As mentioned above, the metabolomics community is working towards the setup of sharing metabolome data, while mining publicly available information and demonstrating the richness of integration of multiple metabolome datasets that remain largely unexplored. At present we are constructing our database, called AtMetExpress112, to store this information. It is freely available and contains detailed information about metabolites detected in Arabidopsis. It has a small and simple GUI tool for performing meta-analyses, allowing easy metabolome meta-analysis for plant biologists using R-Shiny.\n\nPlants produce a diversity of compounds through secondary metabolic pathways. In these secondary compounds, the flavonoids and glucosinolates are useful as herbal medicines to maintain human health. However, a lot of them are still undescribed in public pathway databases. It is therefore important to construct the infrastructure to integrate such metabolites with their pathways in a cross-database manner. Hence, compounds IDs need to be linked rationally for this purpose.\n\nTo address the above challenge, we focused on the following things at BH15. We tried to implement several web applications with R-Shiny to improve visualization tools in our metabolome database, AtMetExpress. To reconstruct secondary metabolite pathway maps on WikiPathways we curated metabolite name, database identifiers of metabolites and reactions (KEGG, KNApSAcK, PlantCyc, and PubChem) in Arabidopsis metabolome data. We focused on flavonoids, which is a well-studied secondary metabolite group in Arabidopsis. We developed the following web applications and tools: a webapp called the Prime Visualization Tool using the R-Shiny framework; an integrated “pathview” Bioconductor package with the Prime Visualization Tool113; an R package for the linkdb RDF client114 to integrate multiple identifiers of major compound databases like PubChem CID, KEGG, and KNApSAcK.\n\nIn addition, we examined the SummarizedExperiment container115 in Bioconductor to use assay, and we discussed the possibility of using the SummarizedExperiment in RDF format. We integrated several Arabidopsis metabolome datasets and partly finished data curations. These curation efforts continue after BH15. Even in the model plant Arabidopsis, the main target of existing large-scale metabolic models was primary metabolism (for example, see 116–119). Our effort to construct curated Arabidopsis flavonoid dataset will help to expand metabolic models of Arabidopsis and lead to a better understanding of the production of flavonoids.\n\nChemical database integration. Small molecules are studied across a broad set of research areas. They are important as a vital component of living systems and are also used in the formulation of pharmaceutical products. Therefore, access to information collected about molecules is key to research and product development. During BH15, we discussed strategies for cooperation between chemical databases. For instance, participants discussed the role of InChIKey120 in their own databases as a primary key for chemical structure. Other discussions focused on increasing interoperability in two ways: First by including additional database cross-references, and second by harmonizing the RDF representation of chemical data. Chemical databases such as PubChem121, Nikkaji122, GlyTouCan123, and the Protein Database Japan (PDBj,124) store data in atomic level formats such as Molfile125, mmCIF126, InChI120, and InChIKey. Participants agreed to use ontologies such as SIO, the Chemical Information ontology (CHEMINF,53), and the Simple Knowledge Organization System (SKOS,127). The RDF data of Nikkaji, KNApSAcK128 and GlyTouCan were modified to use these ontologies. Increased adoption of the ontology-based RDF representation of small molecules will facilitate their integration and reduce the cost of reuse of data from each of the databases.\n\nChemical transformation annotation. We previously developed an ontology for annotating biochemical transformations called Partial Information of chemical transformation (PIERO,129). PIERO provides vocabulary to describe transformations and their attributes along with sets of possible reactions. The vocabulary enables the examination of similar enzymatic reactions, which is particularly important for reactions for which no enzyme has been identified yet. Such reactions are common in secondary metabolism found only in limited organisms. In most cases, they are just putative substrate-product relationships and the reaction equations are not characterized completely. During BH15, we augmented PIERO in a number of ways, including improved RDF interoperability, data curation (adding/correcting more terminology), and reviewing the classification criteria for transformations. One of the most important developments was in the definition of a classification based on reaction characteristics, including the gain or loss of groups, opening or closing the ring structures, intermolecular transfer of groups, formation/digestion of groups, transfer/exchange of groups, and the steps of the reactions.\n\nGlycomics ontology development. Carbohydrates, often referred to as glycans, differ from other biopolymers such as proteins or nucleic acids in the large variety of different building blocks, i.e., monosaccharides, and in the possibility of linking these building blocks in several ways, which often results in branched structures. Furthermore, experimental techniques for glycan identification often yield underdetermined structures with varying degrees of uncertainties. Many providers of glycoinformatics databases and tools have developed individual and non-compatible formats to store all these properties of glycan structures, such as LINUCS130, LinearCode®131, KCF132, GLYDE133, GlycoCT134, or WURCS135. This variety of nomenclature formats is a major reason for a lack of interoperability and data exchange between various glycoinformatics resources136,137. To overcome this situation, development of the glycomics standard ontology (GlycoRDF,138,139) was started during BioHackathon 20124.\n\nGlycoRDF can represent glycan structure information together with literature references or experimental data. MonosaccharideDB140 provides GlycoRDF descriptions of monosaccharides generated from various carbohydrate nomenclature formats. During BH15, participants developed routines to generate GlycoRDF data from WURCS 2.0 nomenclature, which is used by the GlyTouCan structure repository123. Thus, glycomics data can now be retrieved as GlycoRDF from GlyTouCan, GlycoEpitope75, GlycoNAVI141 and WURCS using database guidelines142.\n\nThe group also discussed possible extensions to GlycoRDF that would offer relations between individual monosaccharides. Lactose, for example, is a disaccharide composed of β-D-galactopyranose (1-4)-linked to D-glucose. The latter can be of any ring form or anomeric state due to mutarotation. With relations such as “β-D-glucopyranose is_a D-glucose” or “α-D-glucofuranose is_a D-glucose”, the definition of lactose given above can be used to identify disaccharides with β-D-galactopyranose (1-4)-linked to β-D-glucopyranose or to α-D-glucofuranose as lactose as well. Options to derive such relations from WURCS 2.0 nomenclature have also been discussed. The encoding of these relations in RDF uses existing chemistry definitions such as SIO as much as possible. A first implementation of creating such relations automatically has been added to MonosaccharideDB. The resulting representation will enable (sub-)structure searches with different levels of information in query and target structures, and will also help to assign relations between oligosaccharides.\n\nGlycoinformatics is at the intersection of bioinformatics and chemoinformatics. In the past there have mainly been attempts to establish cross-links between glycan databases and bioinformatics resources, e.g. between UniCarbKB143 and UniProtKB44, which makes sense from the point of view of glycoproteins and protein-carbohydrate complexes. From the small molecules perspective it is coherent to also cross-link with chemoinformatics databases such as PubChem121 or Nikkaji (now subsumed by J-Global,122). Glycan structures cooperation was discussed at BH15. As part of this process several possible formats for data exchange were discussed, such as SMILES144, InChI, mmCIF, WURCS, or mol file. A focus was subsequently put on the conversion of glycan structures to SMILES, and routines to generate SMILES codes from monosaccharide names were developed in a cooperation between PubChem and MonosaccharideDB developers. This will provide an important bridge between glycoinformatics and chemoinformatics and will make it easier for people outside the glycoscience community to access glycomics data. For cooperation between GlyTouCan and PubChem, RDF triples were developed with GlycoRDF, SIO, CHEMINF, DCT, and SKOS.\n\nThe large variety of monosaccharides is mainly caused by the fact that the basic building blocks such as glucose or galactose are often modified by substituents that replace hydrogen atoms or hydroxyl groups, or by introduction of double bonds, deoxy modifications, etc. Currently, no explicit rules exist to define how many modifications can be made to a standard monosaccharide so that it can still be considered as a monosaccharide. Some possible criteria for discrimination between carbohydrate and non-carbohydrate residues were discussed at BH15. We developed a new approach for detecting carbohydrate candidate backbone skeleton. An algorithm for automatic detection of candidate carbon chains of monosaccharide was discussed.\n\n\nResearch methods\n\nOpenLifeData to SADI deployment. The Bio2RDF project145 is now well known within the life sciences LOD community. Recently, OpenLifeData146 completed an effort to provide a distinct view over the Bio2RDF data, with deeper and more rigorous attention to the semantics of the graph, and these views were provided through a distinct set of SPARQL endpoints, with each endpoint acting as a query-rewriter over the original Bio2RDF data147. With these richer and more uniform semantics, it became possible to index each endpoint and automate the construction of SADI Semantic Web Services148 providing discoverable, service-oriented access to all OpenLifeData/Bio2RDF data149—a project that was named OpenLifeData2SADI.\n\nPrior to BH15, the OpenLifeData endpoints were further consolidated into a single endpoint, which caused the OpenLifeData2SADI services to fail. At BH15, the SADI and OpenLifeData project leaders took the opportunity to rewrite the OpenLifeData2SADI automated service deployment codebase. This was originally written as an interdependent mix of Java and Perl scripts, which often took several days to complete. The new codebase is entirely Perl-based, and with the exception of the OpenLifeData indexing step, which is highly dependent on the size of the available OpenLifeData endpoints, runs in less than one hour, deploying tens of thousands of SADI Semantic Web Services over the refactored data. The speed of this new code makes it reasonable to rerun the service deployment dynamically as the underlying OpenLifeData expands or changes, or perhaps automate the re-deployment of services on, for example, a nightly basis. In an ongoing activity since BH15, re-indexing of OpenLifeData has made it possible to capture sample inputs and outputs for each of the resulting SADI services. This information will be added to the SADI service definition documents, allowing for automated service testing and/or more intuitive service registry browser design with, for example, pre-populated “try it now” functionality.\n\nSPARQL query construction. SPARQL150 has emerged as the most widely used query language for RDF datasets. RDF datasets are often provided with web interfaces, called SPARQL endpoints, through which SPARQL queries can be submitted. However, constructing a SPARQL query is a relatively complex task for inexperienced users. SPARQL Builder151 is a web application that assists users in writing SPARQL queries through a graphical user interface. The SPARQL Builder system interactively generates a SPARQL query based on a user-specified path across class-class relationships. At BH15, we worked on the display of candidate paths from metadata, including hierarchical information of the SPARQL endpoint, graphs, classes, properties, class-class relationships, and their statistics, such as the numbers of triples and instances. To be time efficient, we found that it was necessary to pre-compute and store those metadata for fast retrieval. This suggests that it would be ideal that every SPARQL endpoint provides such metadata. We tested our system on datasets drawn from the EBI RDF Platform and Bio2RDF, and our approach could be extended to other RDF datasets. We also developed a prototype152 of a search interface using SPARQL Builder system for 439 datasets contained in the Life Science Database Archive (LSDB Archive,153). The LSDB Archive is a service to collect, preserve and provide databases generated by life sciences researchers in Japan. Using the interface, we can now search for data in the LSDB Archive without knowing the data schema for each dataset.\n\nLODQA integration with DisGeNET and Bio2RDF. LODQA154 is another service being developed to provide a natural language interface to SPARQL endpoints. Users can begin their search with a natural language query, e.g. What genes are associated with Alzheimer’s disease?, from which the system automatically generates corresponding SPARQL queries. LODQA also features a graph editor that allows users to compose queries in a graph representation. While the system is developed to be highly adaptable to any RDF datasets, it does require lexical terms, e.g. labels, of data sets to be pre-indexed.\n\nDuring BH15, we explored the use of the LODQA system with DisGeNET and Bio2RDF. As a result, we found that LODQA could generate effective SPARQL queries for some natural language questions like \"Which genes are involved in calcium binding?\" The LODQA interface to Bio2RDF is publicly available155, while the LODQA interface to DisGeNET is discontinued due to major revisions to DisGeNET.\n\nCrick-Chan query parsing. While LOD and the Semantic Web are rapidly adopted in the biology domain, the majority of biological knowledge is still only available in the form of natural language text, for example in manuscripts on PubMed or in textbooks on the NCBI Bookshelf. The ability to make use of this ocean of data would facilitate knowledge discovery and help bridge the current data retrieval process and the Semantic Web. The success of IBM Watson in the quiz show Jeopardy highlighted the potential of state-of-the-art cognitive computing in answering natural language questions. IBM Watson, however, does not rely so much on semantics or machine learning, but is rather based on queries on unstructured data, with statistical identification of answer domains (Lexical Answer Type). The software for IBM Watson (DeepQA) uses a system to answer a “word” that matches the natural language query by searching through millions of pages of documents, including the entire text of Wikipedia. A scientific fact, or indeed any knowledge, is almost always written in natural language in the form of a manuscript, use of which is relatively less explored in the Semantic Web context. Therefore, at BH15 the G-language Project team aimed to develop a software system, designated “Crick-chan”, that mimics DeepQA to find the most relevant “sentence” (as opposed to a “word” in Watson) from millions of scientific documents. Crick-chan mimics the architecture, and works as follows:\n\n1. The question text first undergoes morphological analysis using Enju156 to extract objective nouns and key verbs. Using a dictionary search, proper nouns are identified.\n\n2. Queries are extended using the Bing search engine (which allows for the largest number of free queries among search engines). At the same time, the question is checked to see whether it belongs to the biology domain.\n\n3. Full text searches are performed for the entire OMIM, PubMed, PubMedCentral, NCBI Bookshelf, Wikipedia, and the entire WWW, via queries to NCBI EUtils and Bing searches.\n\n4. Relevant sentences are extracted from the most relevant matches.\n\n5. Extracted sentences, i.e. the answer hypothesis, are checked for grammatical completeness and are scored according to keywords.\n\n6. Answer confidence is scored according to the data sources and the completeness of key terms.\n\n7. The resulting \"answer\" is presented in a user interface with an artificial character to assist the natural language query process.\n\nFor other general conversation, Crick-chan embeds the AIML bot (ProgramV 0.09) for cases when the question is not considered to belong to the biology domain, and for when there are fewer than two keywords. Crick-chan is publicly accessible157 and it can answer natural language questions such as “What genes are associated with Alzheimer disease?” (Figure 4).\n\nClinical phenotype text mining. Clinical phenotypes, i.e. symptoms and signs, are key for diagnosis and treatment decision-making, particularly for rare or complex disorders158. Delayed or inaccurate diagnosis incurs high economic costs in addition to heavy psychological burden on patients and their families. Deep clinical phenotyping in combination with genotyping are increasingly seen as important components of a vision for precision medicine159. However, vast amounts of phenotypic data available from social media, EHR, biomedical databases, and the scientific literature, are largely inaccessible to direct computation because they are solely available in a narrative form.\n\nNatural Language Processing (NLP) involves the automatic extraction of relevant information from unstructured text and represents it in the form of structured concepts and relationships amenable to further computational analysis. The acquisition of phenotype data is particularly challenging due to the complexity of textual descriptions. Several efforts have explored the extraction of phenotypes from text. For example160, assessed the contribution of feature spaces and training data size on support vector machine model performance for mining phenotypic information on obesity, atherosclerotic cardiovascular disease, hyperlipidemia, hypertension, and diabetes from clinical documents. In the domain of congestive heart failure161, developed automated methods for extracting phenotypic information from clinical documents and from published literature. With the goal of matching phenotypic findings to their correlated anatomical locations as described in clinical discharge summaries162, developed a named entity recognition method based on the Epilepsy and Seizure Ontology (EpSO,163). In fact, a review of studies describing systems or reporting techniques developed for identifying cohorts of patients with specific phenotypes found that 46 out of 97 papers on this topic used techniques based on natural language processing164. In addition, several phenotype-annotated datasets have been recently extracted from journal articles and EHR by using BioNLP and text mining methodologies158,165–169.\n\nThe large-scale acquisition of phenotypic relationships from the literature enable a more complete view on the current knowledge, and thus, more efficient science. The use of text-mined data, i.e. information that is programmatically processed, aggregated and mined, shows much promise for some current challenges such as phenotype definition, hypothesis generation for research, understanding disease and pharmacovigilance. Therefore, their representation as linked data using Semantic Web and LOD approaches and the linking of the annotated literature with the linked data open new avenues for knowledge discovery to advance research and improve health care.\n\nThe curation of biomedical information extracted from scientific publications by text mining is an important current bottleneck for knowledge discovery of new and original solutions for a better health and quality of life. Manual approaches for data curation become more and more time demanding and costly, so that computer assistance in screening (document retrieval) and preparing data (information extraction) is unavoidable. Crowdsourcing approaches have been recently applied with high accuracy170. Therefore, biocuration over the LOD will give a new opportunity to validate knowledge and adding evidence at the same time. The integration of curated and text mined data in the LOD opens new challenges for evidence and provenance tracking. Recent use of the nanopublication approach gives a mechanism for evidence, provenance and attribution tracking171,172.\n\nBH15 offered an opportunity to address different challenges related to the capture and analysis of human phenotype data. The text mining group focused its effort in the primary domains for deep phenotyping: acquisition of phenotype associations from journal articles, integration and alignment of annotation BioNLP tools, evaluation of secondary use of text mining corpora for knowledge discovery, semantic integration of text mined and curated data in the LOD, and curation of text mined data. All these tasks were pursued with a clear emphasis on standardization and interoperability between life sciences databases, text mined datasets and BioNLP tools, with the further aim to linking to the LOD.\n\nNatural language processing of drug effects and indications. Structured drug labels have been used as a source to collect rich representations of drug effects and indications173–175, and these text mined representations have been used in drug repurposing and identification of new targets for known drugs. The Side Effect Resource (SIDER,176) contains a collection of text mined drug effects and indications, using the Unified Medical Language System (UMLS,177) to represent the phenotypes. While the UMLS covers a wide range of clinical signs and symptoms, it does not cover the full set of phenotypes described in non-UMLS biomedical ontologies such as the human Disease Ontology (DO,178) and the Mammalian Phenotype ontology (MP,179).\n\nDuring BH15, we developed an NLP pipeline that identifies the phenotypes occurring in structured drug labels. As vocabularies, we use the phenotype ontologies for mammals, in particular the the Human Phenotype Ontology (HPO,26,166), MP, and the DO. Furthermore, we also use the phenotypic quality ontology (PATO,180), and the Foundational Model of Anatomy (FMA,181), an ontology of human anatomy, as additional vocabularies. Text processing is performed using Lucene, which includes basic text normalization such as stop-word removal and normalization to singular forms. The resulting text-mined annotations of the structured drug labels are freely available182. In the future, these annotations of drugs need to be further evaluated and integrated in linked datasets.\n\nData analysis of text-mined corpora. The combination of high-throughput sequencing and deep clinical phenotyping offers improved capability in pinpointing the underlying genetic etiology of rare disorders. The accuracy of hybrid diagnosis systems is challenged by the vast number of associated variants, many of which lack phenotypic descriptions. At BH15, we sought to learn possible genotype-phenotype relationships from text mining. Specifically, we aimed to use text-mined corpora to learn associations between biological processes disrupted by gene mutations with externalized phenotypes. To do so, we combined two PubMed datasets: i) a dataset generated by the Biomedical Text Mining Group (BTMG) at NIH, comprised of automatically extracted named entities (MeSH terms, genes and mutations); and ii) a second one, generated by the Phenomics team at the Kinghorn Centre for Clinical Genomics (KCCG). The latter covered structured PubMed metadata, i.e., MeSH terms, keywords, etc., as well as HPO annotations. The consolidation of the two datasets, via common MeSH terms, resulted in a final corpus of 6.5M abstracts. To learn biological process – phenotype associations, we added biological process annotations from the Gene Ontology (GO,183). Using the underlying diseases as latent variables (via MeSH terms) and summation as aggregation function, we produced an association matrix between 7,666 HPO terms and 10,438 GO Biological Process terms. The actual use of the matrix has been left for future experiments. Such experiments may cover various aggregation functions (e.g., instead of summation, to use a linear interpolation of the term frequency inverse document frequency (TF-IDF) values of the HPO terms) as well as its application to discovering dense networks of phenotypes – biological processes. The latter could be achieved via some of the following mechanisms:\n\nHierarchical clustering and singular value decomposition (SVD) for ranking HPO - GO BP associations.\n\nPre-clustering of HPO terms based on the HPO top-level abnormalities.\n\nPre-clustering of GO BP terms using higher-level common ancestors.\n\nIntegration of text-mined and curated disease-phenotype data. DisGeNET-RDF contributes to LOD with Gene-Disease Associations (GDAs) obtained from Medline by text mining and integration with associations from different authoritative sources in human genetics184. From release 3.0.0, DisGeNET-RDF also integrates curated Disease-Phenotype Associations (DPAs) to HPO terms for diseases in OMIM, Orphanet, and DECIPHER185 from the HPO project25. In order to examine what are the challenges to integrate text mining with curated DPAs in LOD, we analyzed the DPAs in DisGeNET-RDF (v3.0.0) and the DPAs text-mined from the scientific literature by Hoehndorf et al.167.\n\nHoehndorf2015: This text-mining DPAs dataset contains 6,220 diseases identified by DO identifiers (DOIDs), 9,646 phenotypes identified using the HPO and the MP, and 124,213 DPAs.\n\nHPO2015: This curated DPAs dataset contains 113,203 DPAs between 7,841 diseases and 6,838 phenotypes from OMIM, Orphanet and DECIPHER data sources in which diseases are identified by the corresponding database identifier of provenance, and phenotypes are uniformly identified by HPO identifiers.\n\nWe normalized 6,220 diseases from the Hoehndorf2015 dataset to 5,194 UMLS CUIs by DOID-UMLS cross-references extracted from DO version 2015-06-04 with which only 75% (4,648) of DOIDs can be mapped to UMLS concepts. This is because 17% (1,088) of diseases are described with obsolete DOIDs and 8% (484 DOIDs) do not map to UMLS. Additionally, not all are 1:1 mappings, some N:1 DOID-CUI mappings exist. Therefore, phenotype annotations for different diseases will collapse in a unique UMLS concept.\n\nThe integration of the HPO2015 and Hoehndorf2015 datasets (9,067 and 5,194 UMLS CUIs, respectively) covers 13,596 UMLS concepts of the disease spectrum, of which only 3.2% (665 UMLS CUIs) are in both datasets. This low overlap is due to the fact that each project mainly focuses on covering different disease areas. Whilst the HPO annotation is intended to annotate Mendelian and rare genetic diseases, Hoehndorf et al.’s large-scale literature extraction was focused on broadening the disease class landscape to infectious, environmental, and common diseases. To characterize the disease coverage yielded only by text mining; in Figure 5 we show the top-level DO categories where these novel diseases fall. As can be seen, these novel findings mostly fall in ‘Disease of anatomical entity’ (DOID:7) and ‘disease of cellular proliferation’ (DOID:14566).\n\nIn summary, the analysis of aggregation and integration of text mined and curated disease-phenotype associations in DisGeNET highlights the potential value of text mining in data completeness, annotation, integration, and network biology, which can be used for instance for disease-phenotype ontology construction and curation, knowledge base population, and document annotation. The large-scale integration and publication of text mining DPAs in DisGeNET-RDF opens inference opportunities to grasp potential novel gene-phenotype associations from the current knowledge that promotes our understanding about disease etiology and drug action. However, it is important to keep track of machine-readable provenance and evidence at relationship level for computational analysis and credible knowledge discovery using LOD. Finally, the increase of disease/phenotype terminology and ontology mapping is crucial to foster semantic interoperability and data coverage.\n\nAssessing interoperability of disease terminologies. One benefit of improving the interoperability of disease terminologies is to facilitate translational research and biomedical discovery. Phenotype information is represented using terminologies, vocabularies, and ontologies, but the diverse phenotype spectrum poses serious challenges for their interoperability. For one, phenotypes span from the molecular to the organismal. In addition, while phenotypes in the biological domain are recorded as results from biological experiments, phenotypes in the clinical domain are used to report the state condition of patients186. Furthermore, in current clinical nomenclatures for phenotypes such as MeSH, the 10th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD-10), the nomenclature of the National Cancer Institute (NCI), SNOMED Clinical Terms (SNOMED CT), and UMLS, concepts are covered inconsistently and incompletely186. All these issues affect ontology interoperability, and thus, the quality of their applications. The systematic ontological coding of phenotypic and molecular information in databases and their linking facilitates computational integrative approaches for identifying novel disease-related molecular information187, prioritizing candidate genes for diseases188–191, as well as predicting novel drug-target interactions, drug targets, and indications192,193. The quality of the phenotypic descriptions of a resource will have implications for the quality of their interoperability, and thus, the quality of computational data analyses performed for translational research and knowledge discovery.\n\nIn DisGeNET-RDF (v3.0.0), diseases are normalized with the UMLS CUIs, and are mapped to several disease vocabularies/ontologies with different coverage (see 194 to see disease mapping coverage statistics). Much of the disease data in the data sources of the European Bioinformatics Institute (EBI) is annotated with EFO, such as BioSamples, which aggregates sample information for reference samples and samples used in multi-omics experiments, and the Gene Expression Atlas, which collects gene expression experiments. EFO disease terms have mappings to UMLS, DOID, MeSH, SNOMED CT, OMIM, HPO and ICD-10. EFO also includes and reuses terms from external terminologies such as disease/phenotype terms from the DO, the HPO, and rare disease terms from the Orphanet Rare Disease Ontology (ORDO,195) that include some additional mapping to OMIM and UMLS. In this regard, during BH15, we aimed to increase the integration of DisGeNET and EBI data, by way of its RDF platform.\n\nWe assessed the coverage of EFO concepts against UMLS; from a total of 5,260 terms, only 52 map to the UMLS (see Table 3). Some disease terms do not have cross-references to UMLS concepts. For instance, cancer (EFO_0000311) does not have UMLS CUIs associated, even though it is a general disease term. Nevertheless, the EFO contains over 2000 UMLS mappings from other ontologies, most of them from ORDO, which are manually curated. We suggest that an increase in the mapping between EFO and the UMLS terminologies will benefit data integration and interoperability between RDF datasets such as DisGeNET and other databases that are part of EBI RDF platform.\n\nSemantic haiku generation. Natural language generation is the longstanding problem of generating textual output from textual or non-textual sources196–200. The field has a number of potential applications in the life sciences201–205. One of the projects of BH15 included the construction of a “semantic” haiku generator. Realizing the potential of language generation in communicating information both to scientists and to the public in a way that is acceptable to readers requires the ability to generate text that meets user expectations regarding discourse cohesiveness, genre-appropriate characteristics of word structure, e.g. length, and the like. Poetry generation has been an active area of research in computational linguistics and natural language processing for some time. Here we extend the task definition to the use of LOD, and to the haiku structure, which has not previously been treated in the language generation literature42,206–210. A haiku is a type of poem traditional to Japan; it consists of three verses with five, seven, and five syllables. In light of the work on semantic resources, in particular RDF datasets available through SPARQL, the idea arose to generate a haiku from a SPARQL query by identifying a connected subgraph in which the labels of the resources, or the properties linking them, follow the 5-7-5 syllable pattern of a haiku. Using the CELEX2 dictionary211, which maps English words to their syllables, we wrote a small haiku generator that can be initialized with a SPARQL endpoint and a start node (a resource) from which a search is started to identify a subgraph with the haiku pattern. The prototype code is available at our source code repository62,212. An initial test of the script using the UniProt SPARQL endpoint together with the human Amyloid beta213 protein, which resulted in the following haiku:\n\nAmyloid beta\n\nprotein classified with blood\n\nCoagulation\n\nTo the best of our knowledge, this is the first “semantic” haiku. Although it follows the haiku pattern, additional work is still required to generate haikus that have additional haiku qualities, in particular the occurrence of a word related to one of the four seasons, as tradition requires.\n\nExtending the Common Workflow Language. Computational genomics faces challenges of scalability, reproducibility, and provenance tracking. Larger datasets, such as those produced by The Cancer Genome Atlas214, are now petabyte-sized, while procedures for read mapping, variant calling, genome assembly, and downstream imputation have grown impressively sophisticated, involving numerous steps by various programs. In addition to the need for reproducible, reusable, and trustworthy data, there is also the question of capturing reproducible data analysis, i.e. the steps that happen after raw data retrieval. Genomics analyses involving DNA or RNA sequencing are being used not just for primary research, but now also within the clinic, adding a legal component that makes it essential that analyses can be precisely reproduced. We formed a working group on the challenges of creating pipelines for reproducible data analysis in the context of semantic technologies.\n\nWith the advent of large sequencing efforts, pipelines are getting wider attention in bioinformatics now that biologists regularly have to deal with terabytes of data215. This data can no longer be easily analyzed on single workstations, requiring that analysis is executed on computer clusters and analysis steps are run both serially and in parallel on multiple machines, using numerous software programs. To describe such a complex setup, pipeline runners, or engines, are being developed.\n\nOne key insight from this development is that versioned software is a form of data and can be represented with a unique hash value, e.g., a Secure Hash Algorithm (SHA) value can be calculated over the source code or the binary executables. Also, the steps in a pipeline can be captured in scripts or data and can be represented by a unique hash value, such as calculated by git. This means that the full data analysis can be captured in a single hash value that uniquely identifies a result with the used software and executed analysis steps, together with the raw data.\n\nWe worked on the Common Workflow Language (CWL,216), which abstracts away the underlying platform and describes the workflow in a language that can be used on different computing platforms. To describe the deployed software and make reproducible software installation a reality we also worked on virtualization (Docker) and software packaging and discovery (GNU Guix).\n\nThe CWL is an initiative to describe command line tools and connect them together to create workflows. The original idea of CWL is that a workflow can be described in a ‘document’ and this workflow, once described, can be rerun in different environments. CWL has roots in “make” and similar tools that determine order of execution based on dependency graphs of tasks. Unlike “make”, CWL tasks are isolated and the user must be explicit about its inputs and outputs thereby creating a (hopefully reproducible) document of the workflow. The benefits of explicitness and isolation are flexibility, portability, and scalability: tools and workflows described with CWL can transparently leverage software deployment technologies, such as Docker, be used with CWL implementations from different vendors, and are well suited for describing large-scale workflows in cluster, cloud, and high-performance computing environments where tasks are scheduled in parallel across many nodes.\n\nAt BH15, CWL support was added for the Toil workflow engine217 and work was done on Schema Salad, which is the module used to process YAML CWL files into JSON-LD linked data documents. A tutorial was given on the Common Workflow Language to interested participants. CWL also added the ability to pipe-in JSON objects containing the parameters necessary to run a CWL-wrapped tool218. This allowed CWL to be more easily used with Node.js Streams and thus with the Bionode.io project.\n\nDocker container registry development. One challenge is the creation of standard mechanisms for running tools reproducibly and efficiently. Container solutions, such as Docker, have gained popularity as a solution to this problem. Container technologies have less overhead than full virtual machines (VMs) and are smaller in size. At BH15, we started a registry of bioinformatics Docker containers, which can be used from the CWL, for example. From this meeting evolved the GA4GH Tool Registry API219 that provides ontology-based metadata describing inputs and outputs. Work was also done on an Ensembl API in Docker220.\n\nTo facilitate access to triple stores, we developed a package called Bio-Virtuoso based on Docker. The virtuoso-goloso container runs an instance of the Virtuoso triple store221. This container also receives Turtle, RDF/XML, and OWL format files via the HTTP Post method and internally put them into Virtuoso speedy using the isql command. Graph-feeding containers download data from sources, convert them into RDF if necessary, and send them to virtuoso-goloso. Multiple graph-feeding containers can be combined on demand. To date, we have supported data sources such as the HPO, HPO-annotation, Online Mendelian Inheritance in Man (OMIM,222), OrphaNet223, the HUGO Gene Nomenclature Committee (HGNC,224), OMIM Japanese translation by Gendoo225, and MP179. Bio-Virtuoso is expected to lower barriers to learn SPARQL using real dataset and develop SPARQL-based applications. The project has a GitHub repository226.\n\nGNU Guix extension and deployment. One problem of Docker-based deployment is that it requires special permissions from the Linux kernel, which are not given in many HPC environments. More importantly, Docker binary images are 'opaque', i.e., it is not clear what is inside the container—and its state is affected by what time the container was created and what software is installed, i.e., an intermediate apt-update may generate a different image. Distributing binary images can be considered a security risk—users have to trust the party who created the image227. An alternative to using Docker is using the GNU Guix packaging and deployment system228, which takes a more rigorous approach towards reproducible software deployment. Guix packages, including dependencies, are built from source and generate byte-identical outputs. The hash value of a Guix package is calculated over the source code, the build configuration (inputs), and the dependencies. This means that Guix produces a fully tractable deployment graph that can be regenerated at any time. Guix also supports binary installs and does not require special kernel privileges. As of October 2016, Guix has fast growing support for Perl (473 packages), Python (778), Ruby (153), and R (277). Guix already includes 182 bioinformatics and 136 statistics packages.\n\nAt BH15, we added more bioinformatics packages and documentation229 to GNU Guix and created a deployment of Guix inside a Docker container230. We also packaged CWL in Guix and added support for Ruby gems to Guix which means that existing Ruby packages can easily be deployed in Guix, similar to support for Python packages and R packages. Guix comes with a continuous integration system on a build farm. We want to harvest that information to see when packages are building or failing. See, for example, the Ruby builds231, which contain the SHA values of the package as well as the checkout of the Guix git repository reflecting the exact dependency graph. We are collaborating with Nix and Guix communities to get this information as JSON output so it can be used in a web service.\n\nAssessing the Findable, Accessible, Interoperable, and Reusable Principles. Loosely defined practices in scholarly data publishing prevent researchers from extracting maximum benefit from data intensive research activities, and in some cases make them entirely unusable232. There has been a growing movement encompassing funding agencies, publishers, academics, and the public at large to promote “good data management/stewardship”, and to define and enforce more stringent rules around the publication of digital research objects, including published data, associated software, and workflows, so that they are easily discoverable and readily available for reuse in downstream investigations233. These include international initiatives such as the Research Data Alliance (RDA,234 and235), and Force11236. However, the precise nature and practice of “good data management/stewardship” has largely been up to the producer of digital objects. Therefore, bringing some clarity around the goals and desiderata of good data management and stewardship, and defining simple guideposts to inform those who publish and/or preserve scholarly data, would be of great utility.\n\nStakeholders in the publication of research data, including several authors of this article, participated in the development of an initial draft of the Findable, Accessible, Interoperable, and Reusable (FAIR) principles. The principles were intended to define the key desiderata for the features and/or behaviors that should exist to facilitate data discovery and appropriate scholarly reuse and citation. A public draft237 was published for public comment, and BH15 participants formed a breakout group to carefully examine them against the following criteria: necessity, clarity, conciseness, independence, sufficiency, implementability and relevance. Our critical evaluation led to the development of a revised set of principles that were actionable, and improved coverage and comprehension. The text of these principles was published verbatim in a recent issue of Scientific Data238. These revised principles have been widely lauded239 by researchers240,241, and US and European agencies such as the National Institutes of Health (NIH,242,243) and Elixir244, as being highly informative and providing insight into what it means to be “FAIR”. Future work will focus on the development of quantitative measures of adherence to the principles to assess the FAIRness of a digital resource.\n\nFAIR projector prototype development. Data discovery, integration, and reuse are a pervasive challenge for life sciences research. This is becoming even more acute with the rise of scholarly self-archiving. Much effort has been devoted to the problem of data interoperability, whether through data warehousing245, ontology-based query answering246, or shared application programming interfaces (APIs,247). At BH15, a group of participants further developed a novel idea that was first proposed at a Data FAIRport meeting in 2014, called FAIR Projectors. FAIR Projectors are simple software applications that implement the FAIR principles by “projecting” data in any format (FAIR or non-FAIR) into a FAIR format. A projector will make use of a template-like document called a FAIR Profile, which acts as a meta-schema for the underlying data source. These meta-schemas may be indexed as a means to discover the projection of a dataset that matches the integrative requirements, i.e. the structure and semantics, of a particular workflow.\n\nTo be FAIR themselves, and thus reusable, we have selected the RDF Modeling Language (RML,248), where RDF documents are used to model the structure and semantics of another RDF document. For the functionality of the Projectors, we identified an emergent, RESTful, LOD technology – Triple Pattern Fragments (TPF,1), as a compelling platform that could execute the desired Projector behavior without inventing a new API. This is because TPF natively uses RDF model to publish information which can be served as a RESTful API and thus realizes a Linked Data service by nature. By the end of BH15, we had completed a prototype FAIR Projection system, and had shown how this could be integrated with other components of the nascent FAIR Data publication infrastructure. The result of this development exercise was recently published249.\n\nOntology metadata mapping. Identification of equivalent or similar concepts between vocabularies is key to the analysis of aggregated datasets that use different terminologies. Efforts such as UMLS build and maintain a system for mapping biomedical ontologies to one another. However, such mappings depend on specific versions of the ontologies, and any one version can impact scientific analyses250. Therefore, having access to ontology and mapping metadata is critical to the interpretation and reproducibility of results for bioinformatics research. Initiatives such as the Open Biomedical Ontologies (OBO) Foundry251, Linked Open Vocabularies (LOV,252) and the National Center for Biomedical Ontology’s (NCBO) BioPortal253 have put forward schemas for ontology metadata. The Ontology Metadata Vocabulary (OMV,254) was first published in 2005, but does not reuse current standard vocabularies. In contrast, the Metadata for Ontology Description (MOD,255) does reuse existing properties from SKOS, Friend Of A Friend (FOAF,256) and Dublin Core and Dublin Core Terms (DC, DCT,257).\n\nRecently, the W3C Semantic Web for Health Care and Life Sciences Interest Group258 published a computable specification for the description of datasets, which could also be applied to the description of ontologies259. With respect to mappings and their metadata, SKOS offers a lightweight system for terminology mappings, while Open Pharmacological Concept Triple Store (Open PHACTS,260) put forward a more detailed proposal261 for mappings between RDF datasets, or LinkSets. Yet, in our experience, additional attributes are needed for both ontology and mapping metadata. Therefore, we propose an enhanced metadata scheme as a best practice for ontologies and mappings so as to improve their discovery, analyses, and reporting of results.\n\nOur goal was to define a minimal set of attributes and standards for ontology mapping metadata. We used manually defined and automatically detected disease mappings in DisGeNET262 as a case study263. Our approach involved compiling attributes from the use case, identifying metadata requirements from related initiatives including ontology repositories (Ontobee,264; the Ontology Lookup Service, OLS36; NCBO BioPortal; Aber-OWL,265), large-scale providers of mappings (UMLS, NCBO, Open PHACTS), as well as from individual ontologies including the DO178, HPO26,166, ORDO195, SIO266, the Ontology for Biomedical Investigations (OBI,267) and the Experimental Factor Ontology (EFO,268). We analyzed the mapping metadata and devised a more exhaustive metadata specification for mappings (Table 1) and ontologies (Table 2).\n\nOur work revealed a lack of common annotation in the description of mappings in both the attributes and vocabularies used. The inclusion of justification, provenance, evidence, directionality and versioning of mapping metadata has the potential to increase trust in the interpretation, reliability and reusability of mappings. Other provenance maintaining approaches such as Nanopublications269, singleton properties, or the Ontology of Biomedical AssociatioNs (OBAN,270) that could be used to model this metadata description at individual mapping level to enable a more well detailed and fine-grained semantics description. Having good quality descriptions of ontology and mapping metadata is also relevant for ontology repositories such as BioPortal and Aber-OWL, ontology and data mapping services, and for methods geared towards scientific discovery. The right vocabulary for the metadata description of mappings should be determined through wide community agreement.\n\nExperimental metadata representation. Good science must generate reproducible results271,272, and one aspect of reproducibility is the description of experimental methods and reagents used to generate the reported outcomes. Researchers write the protocols to standardize methods, to share their “know how” with colleagues, and to facilitate the reproducibility of results. Protocols typically specify a sequence of activities that may involve equipment, reagents, critical steps, troubleshooting, tips, and other essential information. Efforts such as CEDAR273 and ISA-Tools274 offer software and data standards to facilitate data collection, management, and reuse of experimental metadata275. Ontologies such as the OBI, the SIO, and the ontology of scientific experiments (EXPO,276) offer vocabulary to capture the design, execution and analysis of scientific experiments, including the protocols, materials used, and the data generated.\n\nThe Experiment ACTions ontology (EXACT,277) suggests a meta-language for the description of experiment actions and their properties. The LABoratory Ontology for Robot Scientists (LABORS,278) that addresses the problem of representing the information required by robots to carry out experiments; LABORS is an extension of EXPO and defines concepts such as “investigation”, “study”, “test”, “trial” and “replicate”. Finally, the SeMAntic RepresenTation for experimental Protocols ontology (SMART Protocols,279) is an application ontology designed to describe an experimental protocol. The SMART Protocol framework proposes a minimal information unit for experimental protocols; the Sample, Instrument, Reagent, Objective model (SIRO, see 279), has been conceived in a way similar to that of the Patient Intervention Comparison Outcome (PICO,280) model. It reuses a number of existing ontologies including the Information Artifact Ontology (IAO,281), the OBI, the BioAssay Ontology (BAO,282), the Chemical Entities of Biological Interest (ChEBI,283), the EFO, the Eagle-i Resource Ontology (ERO,284), EXACT, and the NCBI taxonomy285. Semantic Web technologies including ontologies and Linked Data enable semantic publication of experimental protocols, their classification, and the mining of textual descriptions of experimental protocols.\n\nLimitations of current approaches to experimental metadata include an inability to cover the “digital continuum”—from the highly diverse set of complex processes in laboratories to the needs expressed by regulatory affairs. There also lacks a rapid mechanism to add new concepts into existing ontologies and terminologies. Finally, experimental information is often scattered over a complex network of applications ranging from Laboratory Information Management Systems (LIMS) to text processors and Excel spreadsheets and, most of all, laboratory notebooks. Researchers keep a detailed description of their daily activities, results, problems, plans, derivations of the original plan, ideas, etc. in their laboratory notebooks.\n\nAs a high-level abstraction serving to represent laboratory workflows, we argue, a General Process Model (GPM) is needed. GPMs often represent a networked sequence of activities, objects, transformations, and events that embody strategies for accomplishing a specific task. Such models can be instantiated and specialized as needed. Figure 6 illustrates how a GPM could be further instantiated. The model starts by defining actions in a laboratory. These should be generic so that they can be made concrete as specifics from the laboratory are added, e.g. properties, inputs, and outputs. These generic objects can be linked in terms of inputs and outputs. Once there is an abstract workflow, resources are then allocated. The execution of the workflow instantiates all the properties for each object; data is thus generated with rich, process-related metadata. Repositories such as Dryad286, FigShare287, Dataverse288, and many others structure metadata primarily for describing generic attributes of the datasets while more specialized repositories such as the Gene Expression Omnibus (GEO,289) or the PRoteomics IDEntifications database (PRIDE,290) capture specific elements of the experimental record. Our work to develop a GPM will provide a basis by which published data, metadata, and the experimental protocols used will establish a mechanism by which researchers may execute data sharing plans that meet the expectations of funders, journals and other researchers.\n\nKnowledge graph annotation for human curation. Manual curation of biomedical repositories is a well-established practice in the life sciences domain to improve the accuracy and reliability of data sources. An increasing number of repositories is being made available as networks of concepts and relations, i.e. “knowledge graphs”. Currently, a tool or data source that exposes (part of) a knowledge graph typically provides an annotation facility to allow curators (or the general public) to make or suggest changes. However, such annotations are often only used within the context of that particular tool, for example to notify curators that there may be a problem with a certain data entry, but frequently remain unusable and undiscoverable for other purposes.\n\nFor this reason, we have developed a tool called the Open, Reusable Knowledge graph Annotator (ORKA,291). ORKA is a small, embeddable web service and user interface to capture and publish an annotation event. A typical workflow looks like this:\n\n1. A user or curator of a graph-based resource wants to report a defect or comment on a particular edge of the graph.\n\n2. The resource provides a link that forwards the user to the ORKA user interface.\n\n3. The user is identified by means of one of several open authentication options.\n\n4. The user may now “edit” or comment on the particular graph edge.\n\n5. The annotation is captured and stored and the user will be redirected to the interface of the original resource.\n\nORKA aims to support annotation from a wide range of data sources and tools that are either based on or can be mapped onto a knowledge graph. ORKA can be integrated with such resources by enabling a request to its API. In a user interface this may look like an “Annotate now” link, button, or context menu item, on an association or assertion from the knowledge graph. The API requires minimally a pointer to the original data source and the selected knowledge graph assertion specified as a single triple, i.e. RDF URIs for subject, predicate and object. In subsequent steps, ORKA will collect the identity of the user and record the annotation activity as a self-contained, semantically interoperable digital object.\n\nTo identify the user, we envision a choice from a range of commonly used open authentication identity providers. In the current prototype, Open Researcher and Contributor ID (ORCID) provides the main method of user authentication. We consider the identity of the annotator to be an essential part of the provenance of the annotation: firstly, it can be used to rate or establish trust in the quality of a curator and, secondly, it is needed to reward proper credit to the user for their curation effort.\n\nIn the annotation stage, the user currently has the option to change the relation, add a comment, or both. We found that offering only a limited set of annotation options helps keep the annotation process quick and simple, yet still expressive. Through the selection of an alternative relation, the user suggests an improvement that includes using a more specific predicate or negating the relationship. Relations may be chosen from a pre-loaded set, or from a specific ontology chosen by the user. The free text box can be used to make any additional comments, and currently also serves as a catchall to describe any other type of annotation: for example, to support an assertion with additional evidence, or when a suitable predicate is not readily available.\n\nFinally, ORKA captures the annotation, including provenance information (curator ID, date, original source triple and context), as a semantic digital object using the Nanopublication269 model and the Open Annotation ontology (OA,292). The annotation object is then stored in an annotation repository, which is by default an open Nanopublication store (ORKA can also be reconfigured to store to a private location). Subsequently, the user has the option to browse the repository or return to the original resource from which the annotation request to ORKA was made. Meanwhile, the original data source will receive a notification and link to the annotation object. Data sources may then apply different strategies to incorporate the annotations in their resource: some may first want to perform manual validation, or choose to accept annotations from a selected group of annotators automatically. We note that the semantic description of the annotations and their provenance promotes the reuse of annotations: third parties can access the (public) annotation stores and use them for their own purpose. Attribution can be achieved, as Nanopublications are inherently citable.\n\nWe have designed ORKA as a generic service to annotate different types of graph-based data sources and produce persistent, reusable semantic digital annotation objects. During BH15 we developed a browser bookmarklet that enables annotation of any web page with embedded RDFa statements293. ORKA is currently being developed in the context of the ODEX4All project294 to enable annotation of its core knowledge platform. Initial use cases have suggested a need for additional features, such as annotation of the object of a statement as well as specifying evidence for an annotation (for example by citing published literature). Supporting additional open authentication methods will lower the entry barrier for potential users even further. In the future, we hope to integrate ORKA in other resources and work out scenarios to show how generically reusable annotations result in richer, more accurate data sources and how this helps knowledge discovery in the life sciences domain.\n\n\nConclusions\n\nThe BioHackathon series offers an unparalleled opportunity for scientists and software developers to work together to tackle challenging problems in the life sciences. BH15, the 2015 edition, was no exception, and featured contributions from a wide range of subdisciplines.\n\nOn the topic of semantic metadata, we observed the FAIR principles gaining further traction with the development of additional tooling in the form of FAIR Projectors that represent data in FAIR ways using a template-like system. Likewise pertaining to semantic metadata, work was done at BH15 to assess the state of the art in recording the justification, provenance, evidence, directionality and versioning of ontology mappings. Additionally in this track, participants initiated work on a General Process Model to capture lab experimental metadata as networked sequences of activities, objects, transformations, and events. Lastly in semantic metadata, participants worked on the ORKA system for annotating knowledge graphs by human curators. To contribute to the improvement of reproducibility in bioinformatics, participants in that track worked on three technologies that formally represent the steps of in silico experiments and the computational environment in which such experiments take place. The Common Workflow Language (CWL) is a system to describe command line tools and chain them together. At BH15, participants added CWL support to the Toil workflow engine and worked on CWL components that consume JSON(-LD). In addition, the Docker lightweight system for virtualization (‘containerization’) was targeted at BH15 to enable discovery of bioinformatics containers and simplify deployment of the Virtuoso triple store loaded with bioinformatic data sets. Lastly contributing to reproducibility, participants further extended the GNU Guix ecosystem, an alternative approach for virtualization with certain security advantages, by adding additional bioinformatics packages as well as CWL and Ruby gems to it.\n\nIn the track on genotypes and phenotypes, participants worked on the semantic representation of genotype and phenotype data. This included the modeling of common, stably named and canonically identifiable genomic variation as an RDF graph that was queried using SPARQL. Conceptually related to this, other participants worked on a real-time generated, queryable, semantic representation of VCF data, a commonly used format for representing variant calls such as SNPs. Contributing in this track to the semantic representation of phenotypes, participants worked on the translation of the Human Phenotype Ontology in Japanese. In efforts to contribute to the representation of comparative data within frameworks of shared evolutionary ancestry, participants in the orthology and phylogeny track focused on two challenges. Firstly, work was done on the development of the Orthology Ontology to capture essential concepts pertaining to sequence orthology, including evolutionary events such as sequence duplication and speciation. Secondly, to attempt to place such evolutionary events on absolute time scales, an evaluation was made of the amenability of the FossilCalibrations database on the semantic web by implementing a prototype pipeline that calculates substitution rates for branches between speciation events as a function of time since gene duplication.\n\nContributing to semantic representations in chemistry, participants discussed strategies to advance cooperation between chemical databases, including establishing agreement on which database keys to use, how to make databases more interoperable by denser cross-referencing, and harmonizing RDF representations. Also in this track, more work was done on the PIERO ontology for chemical transformations, including improved RDF interoperability and additional data curation. An important development was the definition of a classification based on reaction characteristics. Moving on to larger molecules, in proteomics, participants assessed the scalability of representing the UniProtKB database in OWL. Other participants in the same track worked on ontologizing proteome data. An important resource in this field is jPOST, for which an assessment of available controlled vocabularies and ontologies took place and work on an RDF schema commenced. In glycomics, participants worked on extending the development of an ontology for representing glycan structures, GlycoRDF, which was initiated at an earlier BioHackathon, in 2012.\n\nIn metabolomics, participants worked on improving the availability on the semantic web of data pertaining to the biochemical analysis of low-molecular-weight metabolites in biological systems. This included a focus on the visualization of plant metabolome profiles and the identification and annotation of metabolites. Participants in this track further worked on the development of visual web applications to expose the metabolome database AtMetExpress.\n\nIn the natural language processing track, participants worked on the capture and analysis of human phenotype data from free form text, i.e. the biomedical literature. Other participants in the same track worked on mining the structured text from drug labels to collect rich representations of drug (side) effects and indications. Also in this track, work was done on data analytics on text-mined corpora, specifically to attempt to learn associations between biological processes disrupted by gene mutations with externalized phenotypes. Large assessments were made of the integration of text mined and curated data and of the interoperability of disease terminology. As a demonstration of the state of the art in generating natural language, a demo was developed that generates a haiku from data on the semantic web.\n\nThe data retrieval and query answering track was concerned with new technologies for interrogating data on the semantic web. This included exposing semantic web services for OpenLifeData through re-implemented, more scalable interfaces. Participants in this track also worked on the SPARQL Builder, a tool for more easily constructing queries in the commonly used, but not very user-friendly, SPARQL language. Other ways to make queries easier included work on LODQA, a system that constructs queries from natural language. A final demo of the state of the art in interrogating the semantic web in a playful way was Crick-Chan, which presents itself through cartoon animations and interacts with users through a chat bot interface.\n\nBH15 thus contributed to many challenges in bioinformatics, including the representation, publication, integration and application of biomedical data and metadata across multiple disciplines including chemistry, biology, genomics, proteomics, glycomics, metabolomics, phylogeny and physiology. A wealth of new semantics-aware applications have been developed through this hackathon that facilitate the reuse of complex biomedical data and build on global efforts to develop an ecosystem of interoperable data and services. As requirements for providing higher quality data and metadata continue to grow worldwide, BioHackathon participants will be well positioned to develop and apply semantic technologies to face the challenges of tomorrow.\n\n\nData availability\n\nNo data are associated with this article.\n\n\nSoftware availability\n\nVersion control repositories to which code was committed during the BioHackathon are aggregated at: https://github.com/dbcls/bh15/wiki/Hackathon-source-code-repositories.\n\nArchived code at time of publication: https://doi.org/10.5281/zenodo.363440562.\n\nLicense: Creative Commons Attribution 4.0 International.", "appendix": "Author contributions\n\n\n\nMD and RAV primarily wrote the manuscript based on the group summaries written by participants. TK, SK, YY, AY, SO, SK2, JK, YW, HW, YK, HO, HB, SK3, SK4, TT organized BH15. All authors attended BH15 and approved the final manuscript.\n\n\nAcknowledgements\n\nBH15 is supported by the Integrated Database Project (Ministry of Education, Culture, Sports, Science and Technology of Japan) and hosted by the National Bioscience Database Center (NBDC) and the Database Center for Life Science (DBCLS). We thank Yuji Kohara, the director of DBCLS, for his support of the BioHackathons.\n\n\nReferences\n\nTriple Pattern Fragments. [cited 2018 May 8]. Reference Source\n\nAntezana E, Kuiper M, Mironov V: Biological knowledge management: the emerging role of the Semantic Web technologies. Brief Bioinform. 2009 [cited 2016 Dec 6]; 10(4): 392–407. PubMed Abstract \| Publisher Full Text\n\nKatayama T, Arakawa K, Nakao M, et al.: The DBCLS BioHackathon: standardization and interoperability for bioinformatics web services and workflows. The DBCLS BioHackathon Consortium*. J Biomed Semantics. 2010; 1(1): 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKatayama T, Wilkinson MD, Aoki-Kinoshita KF, et al.: BioHackathon series in 2011 and 2012: penetration of ontology and linked data in life science domains. J Biomed Semantics. 2014 [cited 2016 Apr 15]; 5(1): 5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKatayama T, Wilkinson MD, Micklem G, et al.: The 3rd DBCLS BioHackathon: improving life science data integration with Semantic Web technologies. J Biomed Semantics. 2013 [cited 2014 Apr 29]; 4(1): 6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKatayama T, Wilkinson MD, Vos R, et al.: The 2nd DBCLS BioHackathon: interoperable bioinformatics Web services for integrated applications. J Biomed Semantics. 2011 [cited 2014 May 15]; 2: 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTopi H, Tucker A: Computing handbook: information systems and information technology. CRC Press/Taylor and Francis; 2014. Reference Source\n\nSilver JK, Binder DS, Zubcevik N, et al.: Healthcare Hackathons Provide Educational and Innovation Opportunities: A Case Study and Best Practice Recommendations. J Med Syst. 2016 [cited 2016 Nov 30]; 40(7): 177. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBusby B, Lesko M; August 2015 and January 2016 Hackathon participants et al.: Closing gaps between open software and public data in a hackathon setting: User-centered software prototyping [version 1; peer review: not peer reviewed]. F1000Res. 2016 [cited 2016 Nov 30]; 5: 672. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCraddock RC, Margulies DS, Bellec P, et al.: Brainhack: a collaborative workshop for the open neuroscience community. Gigascience. 2016 [cited 2016 Dec 3]; 5: 16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMorrison JJ, Hostetter JM, Aggarwal A, et al.: Constructing a Computer-Aided Differential Diagnosis Engine from Open-Source APIs. J Digit Imaging. 2016 [cited 2016 Nov 30]; 29(6): 654–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi LM, Johnson S: Hackathon as a way to raise awareness and foster innovation for stroke. Arq Neuropsiquiatr. 2015 [cited 2016 Nov 30]; 73(12): 1002–4. PubMed Abstract \| Publisher Full Text\n\nSchreiber F, Bader GD, Golebiewski M, et al.: Specifications of Standards in Systems and Synthetic Biology. J Integr Bioinform. 2015 [cited 2016 Nov 30]; 12(2): 258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCeli LA, Ippolito A, Montgomery RA, et al.: Crowdsourcing knowledge discovery and innovations in medicine. J Med Internet Res. 2014 [cited 2016 Nov 30]; 16(9): e216. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDePasse JW, Carroll R, Ippolito A, et al.: Less noise, more hacking: how to deploy principles from MIT’s hacking medicine to accelerate health care. Int J Technol Assess Health Care. 2014 [cited 2016 Nov 30]; 30: 260–4. PubMed Abstract \| Publisher Full Text\n\nVos RA, Biserkov JV, Balech B, et al.: Enriched biodiversity data as a resource and service. Biodivers data J. 2014 [cited 2016 Apr 15]; (2): e1125. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZaaijer S, Columbia University Ubiquitous Genomics 2015 class, Erlich Y: Using mobile sequencers in an academic classroom. eLife. 2016 [cited 2016 Nov 30]; 5: pii: e14258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Bioscience Database Center. [cited 2016 Dec 4]. Reference Source\n\nDatabase Center for Life Science. [cited 2016 Dec 4]. Reference Source\n\nOwen H: Open space technology: a user’s guide. Berrett-Koehler Publishers; 2008. Reference Source\n\nHome \| Global Alliance for Genomics and Health. [cited 2016 Dec 4]. Reference Source\n\nvgteam/vg. [cited 2016 Dec 4]. Reference Source\n\nruby-rdf/rdf-vcf. [cited 2016 Dec 4]. Reference Source\n\nRuby-rdf.github.com by ruby-rdf. [cited 2016 Dec 4]. Reference Source\n\nEclipse RDF4J – formerly known as Sesame. [cited 2016 Dec 4]. Reference Source\n\nKöhler S, Doelken SC, Mungall CJ, et al.: The Human Phenotype Ontology project: linking molecular biology and disease through phenotype data. Nucleic Acids Res. 2014; 42(Database issue): D966–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmitt T, Messina DN, Schreiber F, et al.: Letter to the editor: SeqXML and OrthoXML: standards for sequence and orthology information. Brief Bioinform. 2011 [cited 2016 Apr 18]; 12(5): 485–8. PubMed Abstract \| Publisher Full Text\n\nSonnhammer ELL, Östlund G: InParanoid 8: Orthology analysis between 273 proteomes, mostly eukaryotic. Nucleic Acids Res. 2015; 43(Database issue): D234–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltenhoff AM, Šunca N, Glover N, et al.: The OMA orthology database in 2015: Function predictions, better plant support, synteny view and other improvements. Nucleic Acids Res. 2015; 43(Database issue): D240–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchreiber F, Patricio M, Muffato M, et al.: TreeFam v9: a new website, more species and orthology-on-the-fly. Nucleic Acids Res. 2014; 42(Database issue): D922–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiñarro-Gimenez JA, Madrid M, Fernandez-Breis JT: OGO: an ontological approach for integrating knowledge about orthology. BMC Bioinformatics. 2009; 10 Suppl 10: S13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChiba H, Nishide H, Uchiyama I: Construction of an ortholog database using the semantic web technology for integrative analysis of genomic data. PLoS One. 2015 [cited 2016 Apr 18]; 10(4): e0122802. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTomás Fernández-Breis J, del Carmen Legaz-Garćıa M, Chiba H, et al.: Towards the semantic standardization of orthology content. Reference Source\n\nOrthology Ontology. [cited 2016 Dec 4]. Reference Source\n\nFernández-Breis JT, Chiba H, Legaz-García Mdel C, et al.: The Orthology Ontology: development and applications. J Biomed Semantics. 2016; 7(1): 34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOntology Lookup Service. [cited 2016 Dec 13]. Reference Source\n\nSmith B, Ceusters W, Klagges B, et al.: Relations in biomedical ontologies. Genome Biol. 2005; 6(5): R46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nProsdocimi F, Chisham B, Pontelli E, et al.: Initial implementation of a comparative data analysis ontology. Evol Bioinform Online. 2009 [cited 2016 Apr 18]; 5: 47–66. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSemantic Web Integration Tool (SWIT). [cited 2016 Dec 4]. Reference Source\n\nCarmen Legaz-García MD, Miñarro-Giménez JA, Menárguez-Tortosa M, et al.: Generation of open biomedical datasets through ontology-driven transformation and integration processes. J Biomed Semantics. 2016 [cited 2016 Dec 3]; 7: 32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nqfo/OrthologyOntology. [cited 2016 Dec 4]. Reference Source\n\nGervás P: Engineering Linguistic Creativity: Bird Flight and Jet Planes. 2010; 23–30. Reference Source\n\nSonnhammer EL, Gabaldón T, Sousa da Silva AW, et al.: Big data and other challenges in the quest for orthologs. Bioinformatics. 2014; 30(21): 2993–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUniProt Consortium: UniProt: a hub for protein information. Nucleic Acids Res. 2015 [cited 2016 Apr 15]; 43(Database issue): D204–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNakaya A, Katayama T, Itoh M, et al.: KEGG OC: a large-scale automatic construction of taxonomy-based ortholog clusters. Nucleic Acids Res. 2013; 41(Database issue): D353–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUchiyama I, Mihara M, Nishide H, et al.: MBGD update 2015: Microbial genome database for flexible ortholog analysis utilizing a diverse set of genomic data. Nucleic Acids Res. 2015; 43(Database issue): D270–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPiel WH, Chan L, Dominus MJ, et al.: TreeBASE v. 2: A Database of Phylogenetic Knowledge. E-biosph 2009. London; 2009. Reference Source\n\nLapp H, Bala S, Balhoff JP, et al.: The 2006 NESCent Phyloinformatics Hackathon: A Field Report. Evol Bioinform Online. 2007; 3: 287–96. Publisher Full Text \| Free Full Text\n\nStoltzfus A, Lapp H, Matasci N, et al.: Phylotastic! Making tree-of-life knowledge accessible, reusable and convenient. BMC Bioinformatics. 2013 [cited 2013 Sep 17]; 14: 158. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGernhard T, Ford D, Vos R, et al.: Estimating the relative order of speciation or coalescence events on a given phylogeny. Evol Bioinform Online. 2007 [cited 2016 Apr 15]; 2: 285–93. PubMed Abstract \| 10.1177/117693430600200012Free Full Text\n\nVos RA: Inferring large phylogenies: The big tree problem. Simon Fraser University; 2006 [cited 2017 Mar 6]. Reference Source\n\nVos RA, Mooers AØ: Reconstructing Divergence Times for Supertrees. In: Bininda-Emonds ORP, editor. Phylogenetic supertrees Comb Inf to Reveal Tree Life. Dordrecht: Springer Netherlands; 2004 [cited 2016 Apr 15]. 281–99. Reference Source\n\nNESCent: The National Evolutionary Synthesis Center. [cited 2016 Dec 13]. Reference Source\n\nKsepka DT, Parham JF, Allman JF, et al.: The Fossil Calibration Database-A New Resource for Divergence Dating. Syst Biol. 2015 [cited 2016 Apr 15]; 64(5): 853–9. PubMed Abstract \| Publisher Full Text\n\nFossil Calibration Database. [cited 2016 Dec 4]. Reference Source\n\nVos RA, Caravas J, Hartmann K, et al.: BIO::Phylo-phyloinformatic analysis using perl. BMC Bioinformatics. 2011 [cited 2017 Mar 6]; 12: 63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAntonelli A, Hettling H, Condamine FL, et al.: Toward a Self-Updating Platform for Estimating Rates of Speciation and Migration, Ages, and Relationships of Taxa. Syst Biol. 2017 [cited 2017 Mar 6]; 66(2): 152–166. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSanderson MJ: r8s: inferring absolute rates of molecular evolution and divergence times in the absence of a molecular clock. Bioinformatics. 2003 [cited 2016 Apr 15]; 19(2): 301–2. PubMed Abstract \| Publisher Full Text\n\nOhno S: Evolution by Gene Duplication. New York, New York, USA: Springer New York; 1970. Reference Source\n\nLynch M, Conery JS: The evolutionary fate and consequences of duplicate genes. Science. 2000 [cited 2016 Apr 15]; 290(5494): 1151–5. PubMed Abstract \| Publisher Full Text\n\nParseTTL.groovy. [cited 2016 Dec 4]. Reference Source\n\nVos R, Katayama T: dbcls/bh15: NBDC/DBCLS BioHackathon 2015 (Version v1.0.1). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3634405\n\nRoss PL, Huang YN, Marchese JN, et al.: Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics. 2004 [cited 2016 Apr 18]; 3(12): 1154–69. PubMed Abstract \| Publisher Full Text\n\nOng SE, Blagoev B, Kratchmarova I, et al.: Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics. 2002 [cited 2016 Apr 18]; 1(5): 376–86. PubMed Abstract \| Publisher Full Text\n\nKessner D, Chambers M, Burke R, et al.: ProteoWizard: open source software for rapid proteomics tools development. Bioinformatics. 2008 [cited 2016 Apr 18]; 24(21): 2534–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCox J, Mann M: MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 2008 [cited 2016 Apr 18]; 26(12): 1367–72. PubMed Abstract \| Publisher Full Text\n\nYates A, Akanni W, Amode MR, et al.: Ensembl 2016. Nucleic Acids Res. 2016 [cited 2016 Apr 18]; 44(D1): D710–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPerkins DN, Pappin DJ, Creasy DM, et al.: Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis. 1999 [cited 2016 Apr 18]; 20(18): 3551–67. PubMed Abstract \| Publisher Full Text\n\nCraig R, Beavis RC: TANDEM: matching proteins with tandem mass spectra. Bioinformatics. 2004 [cited 2016 Apr 18]; 20(9): 1466–7. PubMed Abstract \| Publisher Full Text\n\nMayer G, Montecchi-Palazzi L, Ovelleiro D, et al.: The HUPO proteomics standards initiative- mass spectrometry controlled vocabulary. Database. 2013 [cited 2016 Dec 3]; 2013: bat009. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVizcaíno JA, Deutsch EW, Wang R, et al.: ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nat Biotechnol. 2014 [cited 2016 Apr 18]; 32(3): 223–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFarrah T, Deutsch EW, Kreisberg R, et al.: PASSEL: the Peptide Atlas SRM Experiment Library. Proteomics. 2012 [cited 2016 Apr 18]; 12(8): 1170–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWelcome to MassIVE. [cited 2016 Dec 4]. Reference Source\n\njPOSTrepo. [cited 2016 Dec 4]. Reference Source\n\njpost/jpost_pure.owl. [cited 2016 Dec 4]. Reference Source\n\nSaito K, Matsuda F: Metabolomics for functional genomics, systems biology, and biotechnology. Annu Rev Plant Biol. 2010 [cited 2016 Apr 20]; 61: 463–89. PubMed Abstract \| Publisher Full Text\n\nLei Z, Huhman DV, Sumner LW: Mass spectrometry strategies in metabolomics. J Biol Chem. 2011 [cited 2016 Apr 20]; 286(29): 25435–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nErnst M, Silva DB, Silva RR, et al.: Mass spectrometry in plant metabolomics strategies: from analytical platforms to data acquisition and processing. Nat Prod Rep. 2014 [cited 2016 Apr 20]; 31: 784–806. PubMed Abstract \| Publisher Full Text\n\nSumner LW, Lei Z, Nikolau BJ, et al.: Modern plant metabolomics: advanced natural product gene discoveries, improved technologies, and future prospects. Nat Prod Rep. 2015 [cited 2016 Apr 20]; 32: 212–29. PubMed Abstract \| Publisher Full Text\n\nJorge TF, Rodrigues JA, Caldana C, et al.: Mass spectrometry-based plant metabolomics: Metabolite responses to abiotic stress. Mass Spectrom Rev. 2016 [cited 2016 Apr 20]; 35(5): 620–49. PubMed Abstract \| Publisher Full Text\n\nFukushima A, Kusano M: Recent progress in the development of metabolome databases for plant systems biology. Front Plant Sci. 2013 [cited 2016 Apr 20]; 4: 73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSumner LW, Amberg A, Barrett D, et al.: Proposed minimum reporting standards for chemical analysis Chemical Analysis Working Group (CAWG) Metabolomics Standards Initiative (MSI). Metabolomics. 2007 [cited 2016 Apr 20]; 3: 211–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFernie AR, Aharoni A, Willmitzer L, et al.: Recommendations for reporting metabolite data. Plant Cell. 2011 [cited 2016 Apr 20]; 23(7): 2477–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalek RM, Haug K, Conesa P, et al.: The MetaboLights repository: curation challenges in metabolomics. Database (Oxford). 2013 [cited 2016 Apr 20]; 2013: bat029. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarroll AJ, Badger MR, Harvey Millar A: The MetabolomeExpress Project: enabling web-based processing, analysis and transparent dissemination of GC/MS metabolomics datasets. BMC Bioinformatics. 2010 [cited 2016 Apr 20]; 11: 376. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXia J, Wishart DS: MSEA: a web-based tool to identify biologically meaningful patterns in quantitative metabolomic data. Nucleic Acids Res. 2010 [cited 2016 Apr 20]; 38(Web Server issue): W71–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanehisa M, Sato Y, Kawashima M, et al.: KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res. 2016 [cited 2016 Apr 20]; 44(D1): D457–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCerami EG, Gross BE, Demir E, et al.: Pathway Commons, a web resource for biological pathway data. Nucleic Acids Res. 2011 [cited 2016 Apr 20]; 39(Database issue): D685–90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCaspi R, Billington R, Ferrer L, et al.: The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases. Nucleic Acids Res. 2016 [cited 2016 May 2]; 44(D1): D471–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKutmon M, Riutta A, Nunes N, et al.: WikiPathways: capturing the full diversity of pathway knowledge. Nucleic Acids Res. 2016 [cited 2016 May 2]; 44(D1): D488–94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUsadel B, Obayashi T, Mutwil M, et al.: Co-expression tools for plant biology: opportunities for hypothesis generation and caveats. Plant Cell Environ. 2009 [cited 2016 May 2]; 32(12): 1633–51. PubMed Abstract \| Publisher Full Text\n\nFukushima A, Kusano M: A network perspective on nitrogen metabolism from model to crop plants using integrated “omics” approaches. J Exp Bot. 2014 [cited 2016 May 2]; 65(19): 5619–30. PubMed Abstract \| Publisher Full Text\n\nFukushima A, Kanaya S, Nishida K: Integrated network analysis and effective tools in plant systems biology. Front Plant Sci. 2014 [cited 2016 May 2]; 5: 598. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanehisa M, Goto S, Sato Y, et al.: KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res. 2012 [cited 2016 May 2]; 40(Database issue): D109–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVillaveces JM, Jimenez RC, Habermann BH: KEGGViewer, a BioJS component to visualize KEGG Pathways [version 1; peer review: 2 approved]. F1000Res. 2014 [cited 2016 May 2]; 3: 43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKutmon M, van Iersel MP, Bohler A, et al.: PathVisio 3: an extendable pathway analysis toolbox. PLoS Comput Biol. 2015 [cited 2016 May 2]; 11(2): e1004085. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKutmon M, Lotia S, Evelo CT, et al.: WikiPathways App for Cytoscape: Making biological pathways amenable to network analysis and visualization [version 2; peer review: 2 approved]. F1000Res. 2014 [cited 2016 May 2]; 3: 152. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNishida K, Ono K, Kanaya S, et al.: KEGGscape: a Cytoscape app for pathway data integration [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Res. 2014 [cited 2016 May 3]; 3: 144. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKarnovsky A, Weymouth T, Hull T, et al.: Metscape 2 bioinformatics tool for the analysis and visualization of metabolomics and gene expression data. Bioinformatics. 2012 [cited 2016 May 3]; 28(3): 373–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrapov D, Wanichthanarak K, Fiehn O: MetaMapR: pathway independent metabolomic network analysis incorporating unknowns. Bioinformatics. 2015 [cited 2016 May 3]; 31(16): 2757–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXia J, Sinelnikov IV, Han B, et al.: MetaboAnalyst 3.0--making metabolomics more meaningful. Nucleic Acids Res. 2015 [cited 2016 May 3]; 43(W1): W251–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeviumWeb: Dynamic Multivariate Data Analysis and Visualization. [cited 2016 Dec 4]. Reference Source\n\nShiny. [cited 2016 Dec 4]. Reference Source\n\nHorai H, Arita M, Kanaya S, et al.: MassBank: a public repository for sharing mass spectral data for life sciences. J Mass Spectrom. 2010 [cited 2016 May 3]; 45(7): 703–14. PubMed Abstract \| Publisher Full Text\n\nThe Plant/Eukaryotic and Microbial Systems Resource. [cited 2016 Dec 4]. Reference Source\n\nHur M, Campbell AA, Almeida-de-Macedo M, et al.: A global approach to analysis and interpretation of metabolic data for plant natural product discovery. Nat Prod Rep. 2013; 30(4): 565–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGu L, Jones AD, Last RL: LC-MS/MS assay for protein amino acids and metabolically related compounds for large-scale screening of metabolic phenotypes. Anal Chem. 2007 [cited 2016 May 3]; 79(21): 8067–75. PubMed Abstract \| Publisher Full Text\n\nLu Y, Savage LJ, Larson MD, et al.: Chloroplast 2010: a database for large-scale phenotypic screening of Arabidopsis mutants. Plant Physiol. 2011 [cited 2016 May 3]; 155(4): 1589–600. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBell SM, Burgoon LD, Last RL: MIPHENO: data normalization for high throughput metabolite analysis. BMC Bioinformatics. 2012 [cited 2016 May 3]; 13: 10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFukushima A, Kusano M, Mejia RF, et al.: Metabolomic Characterization of Knockout Mutants in Arabidopsis: Development of a Metabolite Profiling Database for Knockout Mutants in Arabidopsis. Plant Physiol. 2014 [cited 2016 May 3]; 165(3): 948–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeKO@PRIMe. [cited 2016 Dec 4]. Reference Source\n\nAtMetExpress@PRIMe. [cited 2016 Dec 4]. Reference Source\n\nLuo W, Brouwer C: Pathview: an R/Bioconductor package for pathway-based data integration and visualization. Bioinformatics. 2013 [cited 2016 Dec 4]; 29(14): 1830–1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nkozo2/linkdbRDF. [cited 2016 Dec 4]. Reference Source\n\nHuber W, Carey VJ, Gentleman R, et al.: Orchestrating high-throughput genomic analysis with Bioconductor. Nat Methods. 2015 [cited 2016 Dec 3]; 12(2): 115–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArnold A, Nikoloski Z: Comprehensive classification and perspective for modelling photorespiratory metabolism. Weber A, editor. Plant Biol (Stuttg). 2013 [cited 2016 Dec 3]; 15(4): 667–75. PubMed Abstract \| Publisher Full Text\n\nde Oliveira Dal’Molin CG, Quek LE, et al.: AraGEM, a Genome-Scale Reconstruction of the Primary Metabolic Network in Arabidopsis. Plant Physiol. 2010 [cited 2016 Dec 3]; 152(2): 579–89. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMintz-Oron S, Meir S, Malitsky S, et al.: Reconstruction of Arabidopsis metabolic network models accounting for subcellular compartmentalization and tissue-specificity. Proc Natl Acad Sci. 2012 [cited 2016 Dec 3]; 109(1): 339–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPoolman MG, Miguet L, Sweetlove LJ, et al.: A Genome-Scale Metabolic Model of Arabidopsis and Some of Its Properties. Plant Physiol. 2009 [cited 2016 Dec 3]; 151(3): 1570–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeller SR, McNaught A, Pletnev I, et al.: InChI, the IUPAC International Chemical Identifier. J Cheminform. 2015 [cited 2016 Nov 30]; 7: 23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim S, Thiessen PA, Bolton EE, et al.: PubChem Substance and Compound databases. Nucleic Acids Res. 2016; 44(D1): D1202–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKunioka T, Miyamura K, Uematsu T, et al.: The development of J-GLOBAL (the formal version): The service design and the feature of J-GLOBAL from a viewpoint of the search action model. J Inf Process Manag. 2012 [cited 2016 Apr 15]; 55(8): 582–90. Publisher Full Text\n\nAoki-Kinoshita K, Agravat S, Aoki NP, et al.: GlyTouCan 1.0--The international glycan structure repository. Nucleic Acids Res. 2016 [cited 2016 Apr 15]; 44(D1): D1237–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKinjo AR, Suzuki H, Yamashita R, et al.: Protein Data Bank Japan (PDBj): maintaining a structural data archive and resource description framework format. Nucleic Acids Res. 2012 [cited 2016 Nov 30]; 40(Database issue): D453–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDalby A, Nourse JG, Hounshell WD, et al.: Description of several chemical structure file formats used by computer programs developed at Molecular Design Limited. J Chem Inf Comput Sci. American Chemical Society; 1992 [cited 2016 Nov 30]; 32(3): 244–55. Publisher Full Text\n\nWestbrook JD, Fitzgerald PM: The PDB format, mmCIF, and other data formats. Methods Biochem Anal. 2003 [cited 2016 Nov 30]; 44: 161–79. PubMed Abstract \| Publisher Full Text\n\nSKOS Simple Knowledge Organization System Reference. [cited 2016 Dec 4]. Reference Source\n\nNakamura Y, Afendi FM, Parvin AK, et al.: KNApSAcK Metabolite Activity Database for retrieving the relationships between metabolites and biological activities. Plant Cell Physiol. 2014 [cited 2016 Apr 20]; 55(1): e7. PubMed Abstract \| Publisher Full Text\n\nKotera M, Nishimura Y, Nakagawa Z, et al.: PIERO ontology for analysis of biochemical transformations: effective implementation of reaction information in the IUBMB enzyme list. J Bioinform Comput Biol. 2014 [cited 2016 Apr 18]; 12(6): 1442001. PubMed Abstract \| Publisher Full Text\n\nBohne-Lang A, Lang E, Förster T, et al.: LINUCS: linear notation for unique description of carbohydrate sequences. Carbohydr Res. 2001 [cited 2016 Apr 15]; 336(1): 1–11. PubMed Abstract \| Publisher Full Text\n\nBanin E, Neuberger Y, Altshuler Y, et al.: A Novel Linear Code Nomenclature for Complex Carbohydrates. Trends Glycosci Glycotechnol. 2002; 14(77): 127–37. Publisher Full Text\n\nAoki KF, Yamaguchi A, Ueda N, et al.: KCaM (KEGG Carbohydrate Matcher): a software tool for analyzing the structures of carbohydrate sugar chains. Nucleic Acids Res. 2004 [cited 2016 Apr 15]; 32(Web Server issue): W267–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSahoo SS, Thomas C, Sheth A, et al.: GLYDE-an expressive XML standard for the representation of glycan structure. Carbohydr Res. 2005 [cited 2016 Apr 15]; 340(18): 2802–7. PubMed Abstract \| Publisher Full Text\n\nHerget S, Ranzinger R, Maass K, et al.: GlycoCT-a unifying sequence format for carbohydrates. Carbohydr Res. 2008 [cited 2016 Apr 15]; 343(12): 2162–71. PubMed Abstract \| Publisher Full Text\n\nTanaka K, Aoki-Kinoshita KF, Kotera M, et al.: WURCS: the Web3 unique representation of carbohydrate structures. J Chem Inf Model. 2014 [cited 2016 Apr 15]; 54(6): 1558–66. PubMed Abstract \| Publisher Full Text\n\nCampbell MP, Ranzinger R, Lütteke T, et al.: Toolboxes for a standardised and systematic study of glycans. BMC Bioinformatics. 2014 [cited 2016 Apr 15]; 15(Suppl 1): S9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLütteke T: Handling and conversion of carbohydrate sequence formats and monosaccharide notation. Methods Mol Biol. 2015 [cited 2016 Apr 15]; 1273: 43–54. PubMed Abstract \| Publisher Full Text\n\nAoki-Kinoshita KF, Bolleman J, Campbell MP, et al.: Introducing glycomics data into the Semantic Web. J Biomed Semantics. 2013 [cited 2016 Apr 15]; 4(1): 39. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRanzinger R, Aoki-Kinoshita KF, Campbell MP, et al.: GlycoRDF: an ontology to standardize glycomics data in RDF. Bioinformatics. 2015 [cited 2016 Apr 15]; 31(6): 919–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMonosaccharideDB. [cited 2016 Dec 7]. Reference Source\n\nGlycoNAVI. [cited 2016 Dec 4]. Reference Source\n\nRDFizingDatabaseGuideline. [cited 2016 Dec 4]. Reference Source\n\nCampbell MP, Peterson R, Mariethoz J, et al.: UniCarbKB: building a knowledge platform for glycoproteomics. Nucleic Acids Res. 2014 [cited 2016 Apr 15]; 42(Database issue): D215–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeininger D: SMILES, a chemical language and information system. 1. Introduction to methodology and encoding rules. J Chem Inf Comput Sci. American Chemical Society. 1988 [cited 2016 Nov 30]; 28(1): 31–6. Publisher Full Text\n\nCallahan A, Cruz-Toledo J, Ansell P, et al.: Bio2RDF Release 2: Improved Coverage, Interoperability and Provenance of Life Science Linked Data. 2013; 200–12. Publisher Full Text\n\nThe openLD group: OpenLifeData - Linked Data for the Life Sciences. 2015.\n\nCallahan A, Cifuentes JJ, Dumontier M: An evidence-based approach to identify aging-related genes in Caenorhabditis elegans. BMC Bioinformatics. 2015; 16: 40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilkinson M, Vandervalk B, McCarthy L: SADI Semantic Web Services - ,cause you can’t always GET what you want! 2009 IEEE Asia-Pacific Serv Comput Conf. IEEE. 2009; 13–8. Publisher Full Text\n\nGonzález AR, Callahan A, Cruz-Toledo J, et al.: Automatically exposing OpenLifeData via SADI semantic Web Services. J Biomed Semantics. 2014; 5: 46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSPARQL 1.1 Overview. [cited 2016 Dec 4]. Reference Source\n\nSPARQL Builder Project. [cited 2016 Dec 4]. Reference Source\n\nSPARQL Builder for DB Archive. [cited 2016 Dec 4]. Reference Source\n\nLSDB Archive. [cited 2016 Dec 4]. Reference Source\n\nLODQA: Question-Answering over Linked Open Data. [cited 2016 Dec 4]. Reference Source\n\nLODQA: Question-Answering over Linked Open Data. [cited 2018 Mar 16]. Reference Source\n\nEnju - An English parser. [cited 2018 Mar 16]. Reference Source\n\nCrick-Chan. [cited 2016 Dec 7]. Reference Source\n\nHuang SH, LePendu P, Iyer SV, et al.: Toward personalizing treatment for depression: predicting diagnosis and severity. J Am Med Inform Assoc. 2014; 21(6): 1069–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobinson PN: Deep phenotyping for precision medicine. Hum Mutat. 2012; 33(5): 777–80. PubMed Abstract \| Publisher Full Text\n\nKotfila C, Uzuner Ö: A systematic comparison of feature space effects on disease classifier performance for phenotype identification of five diseases. J Biomed Inform. 2015 [cited 2016 Dec 3]; 58(Suppl): S92–102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlnazzawi N, Thompson P, Batista-Navarro R, et al.: Using text mining techniques to extract phenotypic information from the PhenoCHF corpus. BMC Med Inform Decis Mak. 2015 [cited 2016 Dec 3]; 15(Suppl 2): S3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCui L, Sahoo SS, Lhatoo SD, et al.: Complex epilepsy phenotype extraction from narrative clinical discharge summaries. J Biomed Inform. 2014 [cited 2016 Dec 3]; 51: 272–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSahoo SS, Lhatoo SD, Gupta DK, et al.: Epilepsy and seizure ontology: towards an epilepsy informatics infrastructure for clinical research and patient care. J Am Med Inform Assoc. 2014; 21(1): 82–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShivade C, Raghavan P, Fosler-Lussier E, et al.: A review of approaches to identifying patient phenotype cohorts using electronic health records. J Am Med Inform Assoc. 2014 [cited 2016 Dec 3]; 21(2): 221–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou X, Menche J, Barabási AL, et al.: Human symptoms-disease network. Nat Commun. 2014; 5: 4212. PubMed Abstract \| Publisher Full Text\n\nGroza T, Köhler S, Moldenhauer D, et al.: The Human Phenotype Ontology: Semantic Unification of Common and Rare Disease. Am J Hum Genet. 2015; 97(1): 111–24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoehndorf R, Schofield PN, Gkoutos GV: Analysis of the human diseasome using phenotype similarity between common, genetic, and infectious diseases. Sci Rep. 2015; 5: 10888. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShah NH: Mining the ultimate phenome repository. Nat Biotechnol. 2013 [cited 2016 Nov 30]; 31(12): 1095–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOellrich A, Collier N, Smedley D, et al.: Generation of silver standard concept annotations from biomedical texts with special relevance to phenotypes. PLoS One. 2015; 10(1): e0116040. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGood BM, Nanis M, Wu C, et al.: Microtask crowdsourcing for disease mention annotation in PubMed abstracts. Pac Symp Biocomput. 2015; 282–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChichester C, Gaudet P, Karch O, et al.: Querying neXtProt nanopublications and their value for insights on sequence variants and tissue expression. J Web Semant. 2014; 29: 3–11. Publisher Full Text\n\nQueralt-Rosinach N, Kuhn T, Chichester C, et al.: Publishing DisGeNET as nanopublications. Brodaric B, editor. Semant Web. IOS Press. 2016 [cited 2016 Dec 3]; 7: 519–28. Publisher Full Text\n\nCampillos M, Kuhn M, Gavin AC, et al.: Drug target identification using side-effect similarity. Science. 2008 [cited 2016 Nov 30]; 321(5886): 263–6. PubMed Abstract \| Publisher Full Text\n\nKuhn M, Campillos M, Letunic I, et al.: A side effect resource to capture phenotypic effects of drugs. Mol Syst Biol. 2010 [cited 2016 Nov 30]; 6: 343. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Q, Deleger L, Lingren T, et al.: Mining FDA drug labels for medical conditions. BMC Med Inform Decis Mak. 2013 [cited 2016 Nov 30]; 13: 53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSIDER Side Effect Resource. [cited 2016 Dec 13]. Reference Source\n\nBodenreider O: The Unified Medical Language System (UMLS): integrating biomedical terminology. Nucleic Acids Res. 2004 [cited 2016 Apr 20]; 32(Database issue): D267–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchriml LM, Arze C, Nadendla S, et al.: Disease Ontology: a backbone for disease semantic integration. Nucleic Acids Res. Oxford University Press. 2012 [cited 2016 Dec 13]; 40(Database issue): D940–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith CL, Goldsmith CA, Eppig JT: The Mammalian Phenotype Ontology as a tool for annotating, analyzing and comparing phenotypic information. Genome Biol. 2004 [cited 2016 Dec 3]; 6(1): R7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPhenotypic Quality Ontology - Summary \| NCBO BioPortal. [cited 2016 Dec 13]. Reference Source\n\nFoundational Model of Anatomy \| Structural Informatics Group. [cited 2016 Dec 13]. Reference Source\n\nIndex of /aber-owl/diseasephenotypes/drugs. [cited 2016 Dec 4]. Reference Source\n\nAshburner M, Ball CA, Blake JA, et al.: Gene Ontology: tool for the unification of biology. Nat Genet. Nature Publishing Group. 2000 [cited 2016 Dec 13]; 25(1): 25–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPiñero J, Queralt-Rosinach N, Bravo A, et al.: DisGeNET: a discovery platform for the dynamical exploration of human diseases and their genes. Database (Oxford). 2015 [cited 2015 Apr 16]; 2015: bav028. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFirth HV, Richards SM, Bevan AP, et al.: DECIPHER: Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources. Am J Hum Genet. 2009; 84(4): 524–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOellrich A, Collier N, Groza T, et al.: The digital revolution in phenotyping. Brief Bioinform. 2016; 17(5) 819–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWashington NL, Haendel MA, Mungall CJ, et al.: Linking human diseases to animal models using ontology-based phenotype annotation. PLoS Biol. 2009; 7(11). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaendel MA, Vasilevsky N, Brush M, et al.: Disease insights through cross-species phenotype comparisons. Mamm Genome. 2015; 26(9–10) 548–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmedley D, Robinson PN: Phenotype-driven strategies for exome prioritization of human Mendelian disease genes. Genome Med. 2015; 7(1): 81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen CK, Mungall CJ, Gkoutos GV, et al.: MouseFinder: Candidate disease genes from mouse phenotype data. Hum Mutat. 2012; 33(5): 858–66. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoehndorf R, Schofield PN, Gkoutos GV: PhenomeNET: a whole-phenome approach to disease gene discovery. Nucleic Acids Res. 2011 [cited 2016 Dec 3]; 39(18): e119. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoehndorf R, Hiebert T, Hardy NW, et al.: Mouse model phenotypes provide information about human drug targets. Bioinformatics. 2014; 30(5): 719–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoehndorf R, Dumontier M, Gkoutos GV: Identifying aberrant pathways through integrated analysis of knowledge in pharmacogenomics. Bioinformatics. 2012; 28(16): 2169–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDisGeNET - a database of gene-disease associations. [cited 2016 Dec 4]. Reference Source\n\nVasant D, Chanas L, Malone J, et al.: ORDO: An Ontology Connecting Rare Disease, Epidemiology and Genetic Data. [cited 2016 Dec 13]. Reference Source\n\nMcDonald DD: Natural Language Generation: An Introduction. University of Massachusetts at Amherst. Department of Computer and Information Science; 1981. Reference Source\n\nMcdonald DD, Pustejovsky JD: Description-Directed Natural Language Generation. 1985; 2: 799–805. Reference Source\n\nSmadja FA, McKeown KR: Automatically extracting and representing collocations for language generation. Proc 28th Annu Meet Assoc Comput Linguist. Morristown, NJ, USA: Association for Computational Linguistics; 1990 [cited 2016 Dec 4]; 252–9. Publisher Full Text\n\nReiter E, Dale R: Building Applied Natural Language Generation Systems. Nat Lang Eng. Cambridge University Press; 1997 [cited 2016 Dec 4]; 3(1): 57–87. Publisher Full Text\n\nReiter E, Dale R: Building natural language generation systems. Cambridge University Press; 2000. Publisher Full Text\n\nPortet F, Reiter E, Hunter J, et al.: Automatic Generation of Textual Summaries from Neonatal Intensive Care Data. Artif Intell Med. Berlin, Heidelberg: Springer Berlin Heidelberg. 2007 [cited 2016 Dec 4]; 227–36. Publisher Full Text\n\nHüske-Kraus D: Suregen-2: a shell system for the generation of clinical documents. Proc tenth Conf Eur chapter Assoc Comput Linguist. - EACL ’03. Morristown, NJ, USA: Association for Computational Linguistics. 2003 [cited 2016 Dec 4]; 2: 215–218. Publisher Full Text\n\nHüske-Kraus D: Text generation in clinical medicine--a review. Methods Inf Med. Schattauer. 2003; 42(1): 51–60. PubMed Abstract \| Publisher Full Text\n\nReiter E, Robertson R, Osman LM: Lessons from a failure: Generating tailored smoking cessation letters. Artif Intell. Elsevier. 2003; 144(1–2): 41–58. Publisher Full Text\n\nHarris DM: Building a large-scale commercial NLG system for an EMR. Proc Fifth Int Nat Lang Gener Conf. Association for Computational Linguistics. 2008; 157–60. Publisher Full Text\n\nAgirrezabal M, Arrieta B, Astigarraga A, et al.: POS-tag based poetry generation with WordNet. 2013; 162–6. Reference Source\n\nFranky: A Rule-based Approach for Karmina Generation. 2013; 24–31. Reference Source\n\nJiang L, Zhou M: Generating Chinese Couplets using a Statistical MT Approach. Manchester. 2008; 377–84. Publisher Full Text\n\nRamakrishnan AA, Devi SL: An alternate approach towards meaningful lyric generation in Tamil. 2010; 31–9. Reference Source\n\nWatanabe K, Matsubayashi Y, Inui K, et al.: Modeling Structural Topic Transitions for Automatic Lyrics Generation. 2014; 422–431. Reference Source\n\nCELEX2 - Linguistic Data Consortium. [cited 2016 Dec 4]. Reference Source\n\nleechuck/semantichaiku. [cited 2016 Dec 4]. Reference Source\n\nAmyloid beta A4 protein. [cited 2016 Dec 4]. Reference Source\n\nCancer Genome Atlas Research Network, Weinstein JN, Collisson EA, et al.: The Cancer Genome Atlas Pan-Cancer analysis project. Nat Genet. 2013 [cited 2016 Apr 18]; 45(10): 1113–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrelles O, Prins P, Snir M, et al.: Big data, but are we ready? Nat Rev Genet. 2011; [cited 2016 Nov 30] 12(3): 224. PubMed Abstract \| Publisher Full Text\n\nCommon Workflow Language. [cited 2016 Dec 4]. Reference Source\n\nBD2KGenomics/toil. [cited 2016 Dec 4]. Reference Source\n\ncwltool-service/cwltool_stream.py. [cited 2016 Dec 4]. Reference Source\n\nga4gh/tool-registry-schemas. [cited 2016 Dec 4]. Reference Source\n\nhelios/ensembl-docker. [cited 2016 Dec 4]. Reference Source\n\nOpenLink Virtuoso Home Page. [cited 2016 Dec 4]. Reference Source\n\nOMIM - Online Mendelian Inheritance in Man. [cited 2016 Dec 13]. Reference Source\n\nOrphanet. [cited 2016 Dec 13]. Reference Source\n\nHGNC database of human gene names. HUGO Gene Nomenclature Committee. [cited 2016 Dec 13]. Reference Source\n\nNakazato T, Ohta T, Bono H: Experimental Design-Based Functional Mining and Characterization of High-Throughput Sequencing Data in the Sequence Read Archive. Aziz RK, editor. PLoS One. 2013 [cited 2016 Dec 3]; 8(10): e77910. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nmisshie/bio-virtuoso. [cited 2016 Dec 4]. Reference Source\n\nCourtès L, Wurmus R: Reproducible and User-Controlled Software Environments in HPC with Guix. Euro-Par 2015: Parallel Processing Workshops. Euro-Par 2015. Lecture Notes in Computer Science.2015; 9523: 579–591. Publisher Full Text\n\nGNU’s advanced distro and transactional package manager — GuixSD. [cited 2016 Dec 4]. Reference Source\n\npjotrp/guix-notes. [cited 2016 Dec 4]. Reference Source\n\nbmpvieira/guix - Docker Hub. [cited 2016 Dec 4]. Reference Source\n\nPackages — GuixSD. [cited 2016 Dec 4]. Reference Source\n\nRoche DG, Kruuk LE, Lanfear R, et al.: Public Data Archiving in Ecology and Evolution: How Well Are We Doing? PLoS Biol. 2015; 13(11): e1002295. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarbers MM, Verschuuren M, de Bruin A: Implementing the European Core Health Indicators (ECHI) in the Netherlands: an overview of data availability. Arch Public Health. 2015 [cited 2016 Apr 15]; 73(1): 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerman F, Wilkinson R, Wood J: Building Global Infrastructure for Data Sharing and Exchange Through the Research Data Alliance. D-Lib Mag. 2014; 20. Publisher Full Text\n\nRDA - Research Data Sharing without barriers. [cited 2016 Dec 4]. Reference Source\n\nMartone ME: FORCE11: Building the Future for Research Communications and e-Scholarship. Bioscience. 2015 [cited 2016 May 2]; 65(7): 635. Publisher Full Text\n\nThe FAIR Data Principles - FOR COMMENT \| FORCE11. [cited 2016 Dec 4]. Reference Source\n\nWilkinson MD, Dumontier M, Aalbersberg IJ, et al.: The FAIR Guiding Principles for scientific data management and stewardship. Sci Data. 2016; 3: 160018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNFU Data4lifesciences \| News \| G20 supports FAIR principles. [cited 2016 Dec 4]. Reference Source\n\nArend D, Junker A, Scholz U, et al.: PGP repository: a plant phenomics and genomics data publication infrastructure. Database (Oxford). 2016 [cited 2016 Dec 3]; 2016: pii: baw033. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRodríguez-Iglesias A, Rodríguez-González A, Irvine AG, et al.: Publishing FAIR Data: An Exemplar Methodology Utilizing PHI-Base. Front Plant Sci. 2016 [cited 2016 Dec 3]; 7: 641. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBourne PE, Lorsch JR, Green ED: Perspective: Sustaining the big-data ecosystem. Nature. 2015 [cited 2016 Apr 18]; 527(7576): S16–7. PubMed Abstract \| Publisher Full Text\n\nBourne PE, Bonazzi V, Dunn M, et al.: The NIH Big Data to Knowledge (BD2K) initiative. J Am Med Informatics Assoc. 2015 [cited 2016 Apr 18]; 22(6): 1114. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIson J, Rapacki K, Ménager H, et al.: Tools and data services registry: a community effort to document bioinformatics resources. Nucleic Acids Res. 2016 [cited 2016 Apr 18]; 44(D1): D38–47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAntezana E, Blondé W, Egaña M, et al.: BioGateway: a semantic systems biology tool for the life sciences. BMC Bioinformatics. BioMed Central. 2009 [cited 2016 May 2]; 10 Suppl 10: S11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCallahan A, Cruz-Toledo J, Dumontier M: Ontology-Based Querying with Bio2RDF's Linked Open Data. J Biomed Semantics. 2013; 4 Suppl 1: S1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRahimzadeh V, Dyke SO, Knoppers BM: An International Framework for Data Sharing: Moving Forward with the Global Alliance for Genomics and Health. Biopreserv Biobank. 2016 [cited 2016 May 2]; 14(3): 256–95. PubMed Abstract \| Publisher Full Text\n\nDimou A, Vander Sande M, Colpaert P, et al.: RML: A Generic Language for Integrated RDF Mappings of Heterogeneous Data. Proc 7th Work Linked Data Web. 2014 [cited 2016 Dec 4]. Reference Source\n\nWilkinson MD, Verborgh R, Santos LOB da S, et al.: Interoperability and FAIRness through a novel combination of Web technologies. PeerJ Inc. 2017. Publisher Full Text\n\nClarke EL, Loguercio S, Good BM, et al.: A task-based approach for Gene Ontology evaluation. J Biomed Semantics. 2013 [cited 2016 Nov 30]; 4 Suppl 1: S4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith B, Ashburner M, Rosse C, et al.: The OBO Foundry: coordinated evolution of ontologies to support biomedical data integration. Nat Biotechnol. 2007 [cited 2016 May 2]; 25(11): 1251– 5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLinked Open Vocabularies. [cited 2016 Dec 4]. Reference Source\n\nWhetzel PL, Noy NF, Shah NH, et al.: BioPortal: enhanced functionality via new Web services from the National Center for Biomedical Ontology to access and use ontologies in software applications. Nucleic Acids Res. 2011; [cited 2016 May 2] 39: W541–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHartmann J, Palma R, Sure Y, et al.: Ontology Metadata Vocabulary and Applications. Springer Berlin Heidelberg; 2005; [cited 2016 Dec 13] 906–15. Publisher Full Text\n\nDutta B, Nandini D, Engineering BCR: MOD: Metadata for Ontology Description and Publication. 2015. Reference Source\n\nGraves M, Constabaris A, Brickley D: FOAF: Connecting People on the Semantic Web. Cat Classif Q. Taylor & Francis Group. 2007 [cited 2016 Dec 13]; 43(3–4): 191–202. Publisher Full Text\n\nWeibel S: The Dublin Core: A Simple Content Description Model for Electronic Resources. Bull Am Soc Inf Sci Technol. Wiley Subscription Services, Inc., A Wiley Company. 2005 [cited 2016 Dec 13]; 24(1): 9–11. Publisher Full Text\n\nSemantic Web Health Care and Life Sciences Interest Group. [cited 2016 Dec 13]. Reference Source\n\nDumontier M, Gray AJG, Marshall MS, et al.: The health care and life sciences community profile for dataset descriptions. PeerJ. 2016 [cited 2016 Nov 30]; 4: e2331. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliams AJ, Harland L, Groth P, et al.: Open PHACTS: semantic interoperability for drug discovery. Drug Discov Today. 2012; 17(21–22): 1188–98. PubMed Abstract \| Publisher Full Text\n\nDataset Descriptions for the Open Pharmacological Space. [cited 2016 Dec 4]. Reference Source\n\nBauer-Mehren A, Rautschka M, Sanz F, et al.: DisGeNET: a Cytoscape plugin to visualize, integrate, search and analyze gene-disease networks. Bioinformatics. Oxford University Press. 2010 [cited 2016 Dec 13]; 26(22): 2924–6. PubMed Abstract \| Publisher Full Text\n\nQueralt-Rosinach N, Piñero J, Bravo À, et al.: DisGeNET-RDF: harnessing the innovative power of the Semantic Web to explore the genetic basis of diseases. Bioinformatics. 2016; 32(14): 2236–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXiang Z, Mungall C, Ruttenberg A, et al.: Ontobee: A Linked Data Server and Browser for Ontology Terms. Reference Source\n\nHoehndorf R, Slater L, Schofield PN, et al.: Aber-OWL: a framework for ontology-based data access in biology. BMC Bioinformatics. 2015 [cited 2016 Dec 13]; 16: 26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDumontier M, Baker CJ, Baran J, et al.: The Semanticscience Integrated Ontology (SIO) for biomedical research and knowledge discovery. J Biomed Semantics. 2014 [cited 2016 Apr 15]; 5(1): 14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBandrowski A, Brinkman R, Brochhausen M, et al.: The Ontology for Biomedical Investigations. PLoS One. 2016 [cited 2016 May 2]; 11(4): e0154556. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalone J, Holloway E, Adamusiak T, et al.: Modeling sample variables with an Experimental Factor Ontology. Bioinformatics. 2010 [cited 2016 May 2]; 26(8): 1112–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nnanopub.org. [cited 2016 Dec 4]. Reference Source\n\nSarntivijai S, Vasant D, Jupp S, et al.: Linking rare and common disease: mapping clinical disease-phenotypes to ontologies in therapeutic target validation. J Biomed Semantics. 2016 [cited 2016 Dec 13]; 7: 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBegley CG, Ioannidis JP: Reproducibility in science: improving the standard for basic and preclinical research. Circ Res. 2015; 116(1): 116–26. PubMed Abstract \| Publisher Full Text\n\nMesirov JP: Computer science. Accessible reproducible research. Science. 2010 [cited 2016 Apr 15]; 327(5964): 415–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMusen MA, Bean CA, Cheung KH, et al.: The center for expanded data annotation and retrieval. J Am Med Inform Assoc. 2015; 22(6): 1148–52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRocca-Serra P, Brandizi M, Maguire E, et al.: ISA software suite: supporting standards-compliant experimental annotation and enabling curation at the community level. Bioinformatics. Oxford University Press. 2010 [cited 2016 Dec 13]; 26(18): 2354–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRocca-Serra P, Salek RM, Arita M, et al.: Data standards can boost metabolomics research, and if there is a will, there is a way. Metabolomics. 2016 [cited 2016 May 2]; 12: 14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSoldatova LN, King RD: An ontology of scientific experiments. J R Soc Interface. The Royal Society. 2006 [cited 2016 May 3]; 3(11): 795–803. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSoldatova LN, Aubrey W, King RD, et al.: The EXACT description of biomedical protocols. Bioinformatics. Oxford University Press. 2008 [cited 2016 Dec 3]; 24(13): i295–303. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKing RD, Liakata M, Lu C, et al.: On the formalization and reuse of scientific research. J R Soc Interface. The Royal Society. 2011 [cited 2016 May 3]; 8(63): 1440–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGiraldo O, García A, Corcho O: SMART Protocols: SeMAntic RepresenTation for Experimental Protocols. Linked Sci 2014—Mak Sense Out Data. 2014 [cited 2016 May 3]. Publisher Full Text\n\nAslam S, Emmanuel P: Formulating a researchable question: A critical step for facilitating good clinical research. Indian J Sex Transm Dis AIDS. Medknow Publications. 2010 [cited 2016 May 3]; 31(1): 47–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\ninformation-artifact-ontology/IAO. [cited 2016 Dec 13]. Reference Source\n\nVisser U, Abeyruwan S, Vempati U, et al.: BioAssay Ontology (BAO): a semantic description of bioassays and high-throughput screening results. BMC Bioinformatics. 2011 [cited 2016 May 2]; 12: 257. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDegtyarenko K, de Matos P, Ennis M, et al.: ChEBI: a database and ontology for chemical entities of biological interest. Nucleic Acids Res. 2008 [cited 2016 May 2]; 36(Database issue): D344–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEagle-I Research Resource Ontology - Summary \| NCBO BioPortal. [cited 2016 Dec 13]. Reference Source\n\nHome - Taxonomy - NCBI. [cited 2016 Dec 4]. Reference Source\n\nDryad Digital Repository - Dryad. [cited 2016 Dec 4]. Reference Source\n\nfigshare - credit for all your research. [cited 2016 Dec 4]. Reference Source\n\nThe Dataverse Project - Dataverse.org. [cited 2016 Dec 4]. Reference Source\n\nHome - GEO - NCBI. [cited 2016 Dec 13]. Reference Source\n\nMartens L, Hermjakob H, Jones P, et al.: PRIDE: the proteomics identifications database. Proteomics. 2005 [cited 2016 Apr 18]; 5(13): 3537–45. PubMed Abstract \| Publisher Full Text\n\nORKA - Open, Reusable Knowledge graph Annotator - ORKA - Confluence. [cited 2017 Mar 3]. Reference Source\n\nWeb Annotation Vocabulary. [cited 2016 Dec 4]. Reference Source\n\nRDFa. [cited 2016 Dec 4]. Reference Source\n\nODEX4All. [cited 2016 Dec 4]. Reference Source" }	[ { "id": "60449", "date": "31 Mar 2020", "name": "Jeremy G. Frey", "expertise": [ "Reviewer Expertise Chemical Informatics", "Physical Chemistry", "Digital Economy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written report of a Hackathon activity in the area of semantics and ontologies for life sciences with a view to using these semantic technologies and knowledge structures to enhance the reproducibility and I would say the use and re-use of life sciences data and methods in a number of typically gene and protein related areas. While the report is on an activity which I presume took place in 2015 the material is very relevant and not dated.\n\nThe report contains very good summaries of the prior art on the areas, especially ontologies that cover the areas of interest and are of potential use across the whole of the scientific research life cycle. The details of the challenges, data made available, teams and goals are provided and will benefit the community though it will need detailed reading to absorb the extensive material provided by the authors. The extensive references are themselves a very useful research source.\nI consider this to be a well written and useful contribution to the literature which will help the community in building new and useable systems (and also not to re-invent things that do already work).\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "60451", "date": "07 Apr 2020", "name": "James P. Balhoff", "expertise": [ "Reviewer Expertise semantic technologies", "bio-ontologies", "bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a sprawling and fascinating report from a hackathon in 2015. The activities involve development of software and standards across a broad range of subdisciplines within the life sciences, but are united by largely basing their approaches on the application of semantic technologies. As such, the paper is an excellent overview of a wide variety of ways in which semantic technologies are being employed with great success in the biological sciences. Areas targeted include genotype/phenotype data, orthology and phylogeny, proteomics, metabolomics, data retrieval and querying, natural language processing, reproducibility, and metadata representation. Despite the several years that have elapsed since then, many of the tools built and insights gleaned still have relevance today, and so we are grateful that this work has been written and published. However, we believe that this five year gap between event and publication provides a valuable opportunity for the authors to reflect on how that hackathon work was useful, both in its original context as well as today. We think the authors did an excellent job describing this in the section “Assessing the Findable, Accessible, Interoperable, and Reusable Principles”, but some other sections suffer by not being clear about what was developed before, during and after the hackathon. It may turn out that some of this work was not particularly useful in the long term — which is only to be expected in a hackathon — but it might also be that a good idea from five years ago has been subsequently overlooked, and this work might be in a position to call attention to such ideas.\n\nHaving explicit subsections entitled “Changes in the landscape since 2015” in every section could be helpful in making this clearer. There are also several references to events that were contemporaneous with the hackathon; for example, in the section “Molecular evolutionary process calibration”, the authors write that “Recently, a working group at the National Evolutionary Synthesis Center (NESCent) …”. However, this is incorrect to state in a document published in 2020, as NESCent was shut down in June 2015. Clarifying which parts of the text are true as of the workshop and which are true today would help to prevent such errors.\nUnderstandably for activities at a hackathon, some activities could be described in a little more detail, while some descriptions could be more concise. Some notes on the various descriptions follow:\nVariation graph construction:\nIt would be helpful to state which triplestore database was used when stating performance results.\n\nOrthology ontology development and application:\nGrammatical issue in sentence including “Although the standard mapping and transformation by SWIT was largely able to transform the content of the three databases, though a few resource-specific rules were necessary because” (remove ‘though’?).\n\nMolecular evolutionary process calibration:\nThe authors point out that a particular JSON resource is “convenient for programmers but it also means that certain concepts used in the JSON are ambiguous as they are not linked to any controlled vocabulary or ontology.” They should mention JSON-LD as a possible solution to this problem, a technology they refer to several times elsewhere in the paper.\n\nProtein semantic representation:\nIt would be useful to include a brief statement about how the particular axiomatization described supports some use cases. Besides OWL 2 EL scalability, what motivated this particular design? \"For complex datasets, the additional semantics of OWL, which includes assertions of disjointness, i.e. the explicit semantic distinction between classes and their instances, and axioms restricting the use of classes and object properties, may be particularly beneficial.\" Here, it seems like an inaccurate definition is provided for ‘disjointness’ (“explicit semantic distinction between classes and their instance”). This is not what disjointness means, but perhaps this was just meant to be a list of logical features provided by OWL. Remove ‘i.e.’? A layout issue we noted was the sentence “We also generate the following axioms (here expressed in Manchester OWL Syntax)”, which should not be a bullet point.\n\nTools for metabolite identification and interpretation / Plant metabolome database development:\nThese sections provide a large amount of background information, taking longer to reach the description of what was accomplished at the hackathon.\n\nChemical database integration:\nPerhaps it wasn't discussed at the hackathon, but we are curious how the ChEBI ontology fits into this picture of harmonization across chemical databases.\n\nClinical phenotype text mining:\nThe superscript citation format makes some of the sentences oddly worded (e.g., second paragraph) where it seems like the author name is meant to be in the sentence. \"For example160, assessed the contribution of …”. In this case, it appears that the citation was intended to be included inline, i.e. “For example, Kotfila and Uzuner (2005) assessed ...”. In these cases, the text should be rewritten so that it is easier to read.\n\nThe abbreviation section could benefit from hyperlinks to the ontologies (and possibly also the organizations) being linked to.\nApart from these relatively minor issues, we are grateful that the authors have published this work and recorded the activities at what appears to be a wide-ranging and productive hackathon. Also, congratulations to them on producing the first semantic haiku!\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-136
https://f1000research.com/articles/8-2148/v1	30 Dec 19	{ "type": "Study Protocol", "title": "The Adolescent Knee Pain (AK-Pain) prognostic tool: protocol for a prospective cohort study", "authors": [ "Alessandro Andreucci", "Sinead Holden", "Martin Bach Jensen", "Michael Skovdal Rathleff", "Sinead Holden", "Martin Bach Jensen", "Michael Skovdal Rathleff" ], "abstract": "Background: One in three children and adolescents experience knee pain. Approximately one in two adolescents with knee pain will continue to experience pain even five years later and have low quality of life. The general practitioner (GP) is the first point of contact for children and adolescents with knee pain in Denmark. There is a variety of treatments being delivered in general practice, despite similar symptoms and patients’ characteristics. This suggests a need to support the GPs in identifying those at high risk of a poor outcome early on, in order to better allocate resources. The aim of this study is to develop a user-friendly prognostic tool to support GPs’ management of children and adolescents’ knee pain. Methods: A preliminary set of items in the prognostic tool were identified using systematic reviews and meta-analysis of individual participant data. Following feedback from GPs and children and adolescents on the content and understanding, the tool was piloted and implemented in general practice. A cohort of approximately 300 children and adolescents (age 8-19 years old) is being recruited from general practices (recruitment period, July 2019 – June 2020). Clinically meaningful risk groups (e.g. low/medium/high) for the recurrence/persistence of knee pain (at 3 and 6 months) will be identified. Discussion: If successful, this prognostic tool will allow GPs to gain insights into the likely prognosis of adolescents with knee pain and subsequently provide the first building blocks towards stratified care, where treatments will be matched to the patients’ prognostic profile. This has the potential to improve the recovery of children and adolescents from knee pain, to improve the allocation of resources in primary care, and to avoid the decline in physical activity and potential associated health and social consequences due to adolescent knee pain. Registration: Registered with ClinicalTrials.gov on 24 June 2019 (ID NCT03995771).", "keywords": [ "Prognosis", "Prognostic factor", "Adolescent", "Knee pain" ], "content": "Background\n\nOne in three adolescents experience knee pain1. Knee pain is associated with low quality of life and lower sporting ability compared to adolescents without knee pain1. In addition to the impact on the individual adolescents, knee pain has an impact on their family2 and an economic impact, due to both direct (e.g. primary care visits, community services use, medication use) and indirect (e.g. parental productivity work loss and days off work) costs3,4. Adolescent knee pain (AKP) was once thought to be innocuous and self-limiting, but new data has challenged this assumption5. A recent prospective cohort study demonstrated that 40% of adolescents with knee pain still experienced knee pain even after five years5. Knee pain is linked to both health and social consequences5,6. Children and adolescents with knee pain are likely to reduce their sport participation, which may have implications for overall health in later life (e.g. higher adiposity, impaired sleep)1,7–10. For some adolescents it results in school absence8,11 and for one in seven it affects their choice of job or career5,12.\n\nWhile there is a large body of knowledge on adult knee pain13–15, less is known in children and adolescents7,16. Potential prognostic factors for a poor outcome in AKP include female sex, high leisure time sport participation, low health-related quality of life, high baseline frequency of knee pain6. These preliminary prognostic factors have been identified from single studies and have never been replicated in independent cohorts. Therefore, there is a need to further test and replicate these prognostic factors in other studies in order to confirm this preliminary evidence, also in contexts such as primary care.\n\nChildren and adolescents with knee pain commonly consult their general practitioner (GP), who is their first point of contact and who discuss with them and their parents the different treatment options (e.g. referral to a specialist, education on how to manage knee pain, or exercises). This is largely based on clinical experience as there is a lack of clinical practice guidelines and original research on the management of AKP. This may result in heterogenous care, unnecessary over-medicalization17,18 and large differences in treatments, despite presenting with similar symptoms and characteristics.\n\nOne potential option to support clinical decision making is to develop decision aids such as prognostic tools. These tools often consist of items with prognostic value that can be asked during the clinical consultation or completed prior to the consultation, and can be used to stratify patients depending on their prognosis19. Examples of prognostic tools that have already been developed include the Keele STarT Back Screening Tool (SBST) for lower back pain in adults20 or the Pediatric Pain Screening Tool (PPST) for general pediatric pain19. However, a prognostic tool to be used specifically for the prognosis of AKP in primary care has not been developed yet. The development of a prognostic tool for AKP would fill this gap and provide supporting information to guide GPs in their clinical decision towards a stratified care based on the category of risk for AKP (derived from the patients’ individual characteristics).\n\n\nMethods\n\nThe aim of this study is to develop and test a prognostic tool for AKP to be used in general practice.\n\nThis study is designed as a prospective cohort study and the protocol follow The Recommendations for Interventional Trials (SPIRIT) 2013; the completed checklist can be accessed at the Harvard Dataverse online repository21.\n\nParticipants will be recruited from general practices in Denmark, with data collected from July 2019 until June 2020.\n\nThe recruitment strategy for the project has been developed together with experienced GPs and researchers within the field of general practice to ensure feasibility of recruitment and high generalisability. The recruitment includes two stages; the recruitment of GPs and the recruitment of participants.\n\nGPs working in the North Denmark Region and Odense area in Denmark are eligible for inclusion. These areas were chosen for logistic reasons, but more regions (e.g. Central Denmark Region, Copenhagen area) may be added if recruitment is slower than anticipated. To obtain enough GPs involved in the study, efforts will be done to comply with the Solberg’s seven R-factors for recruiting medical groups for research (i.e. relationship, reputation, requirements, reward, reciprocity, resolute behaviour and respect)22. First, contact will be made with GPs in order to ask them their availability to join the research. The first contact will be brief, but informative. Second, introductory meetings will be held with GPs and their staff (i.e. secretaries and nurses) to confer the importance, contents and goals of the study, information regarding the participants’ eligibility criteria and to plan the recruitment of participants. After the beginning of participant recruitment, regular meetings with the GPs and their staff will be held during the follow-up in order to monitor the recruitment rate of participants and support them in recruitment (e.g. if changes are needed to the strategy used at their clinic).\n\nChildren and adolescents who consult their GP because of knee pain (of both traumatic and non-traumatic origin) during a period of recruitment of at least 6 months (start in July 2019) are eligible for inclusion. Children and adolescents will have to be between 8 and 19 years old. The age of 8 is considered to be the lowest age for the children to be able to complete a pain questionnaire or a pain chart without adult guidance23 (provided that the questions are properly worded by taking into account the age-related cognitive abilities24,25), and the age of 19 is defined as the upper limit for the period of adolescence by the World Health Organization26. The following exclusion criteria will be assessed and applied by the person in charge of recruiting the participants (i.e. either the GP or a member of the staff):\n\nAge below 8 years old or over 19 years old\n\nConsultation for musculoskeletal pain only in a body region different from the knee\n\nPain originated from specific non-musculoskeletal conditions (e.g. cancer, infections)\n\nChild is vulnerable (e.g. he/she has experienced a recent trauma and the distress may have an impact on the self-report making it not valid)24\n\nInability to take part to the study because of inability to understand the questionnaire\n\nParticipants will be recruited from general practices on the basis of consultation for knee pain. The study will be introduced to both parents and children before the consultation by a person working at the general practice (e.g. the GP/nurse). If patients meet the inclusion criteria for being eligible into the study (e.g. patient aged between 8 and 19 years old who consult for knee pain) and agree to participate, they will be provided with the study material (i.e. prognostic tool to be completed). In the primary stages of the study the person in charge of recruiting participants will be assisted in recruitment by the primary investigator of this study (A.A.) or by a research assistant who will attend the participating general practices.\n\nComplementary recruitment strategies that might be applied to maximize the recruitment rate are the advertisement of the study through social media (e.g. Facebook, Twitter, Snapchat) and through explicative posters displayed at the participating general practices. In this case, it will be reminded that the premise for participation is that participants have been seen by their GP and knee pain was part of the consultation. These complementary strategies will be applied if less than approximately 100 participants in 3 months will be recruited.\n\nTo encourage participation, children and adolescents will be offered two cinema tickets to take part to the study. The first ticket will be given at baseline, when children and adolescents decide to take part in the study and return the questionnaire. The second ticket will be given when the participants have completed the follow-up (e.g. provided information at both the 3-month and 6-month follow-up points).\n\nData will be collected through a questionnaire delivered at the general practice to children and adolescents who consult for knee pain (available as Extended data27). The questionnaire will be either paper-based or collected with the support of a tablet through a link to the REDcap web application for online surveys28 depending on the GPs’ preference for data collection. A further recruitment through social media might be applied to complement the baseline collection of data, with data collected through a questionnaire home-mailed or delivered with an e-mail with a link to the web application for online surveys.\n\nOutcomes will be collected by questionnaires by a combination of self-reported or parent-reported (through an e-mail with a link to the web application for online surveys or text message) at follow-up (two time points: 3-month and 6-month follow-up). The questionnaires will include questions about pain characteristics (e.g. severity of pain, period free of knee pain, disability and activity limitations due to pain29,30) taken from previously validated pain questionnaires or pain scales, and about who is the person replying to the questionnaire (child alone/parent alone/child together with the parent). In order to limit loss to follow-up, an e-mail/text message reminder will be sent to participants if they do not complete the follow-up questionnaire within one week from the day when they are supposed to reply (i.e. 3-month and 6-month follow-up). A second reminder will be sent one week after the first reminder if they still will not have completed the follow-up questionnaire. Reasons for loss to follow-up will be assessed by contacting through a phone call/e-mail/text message those participants who do not reply to 3 consecutive follow-up reminders and asking about reasons for leaving the study.\n\nExamples of potential participants’ timeline (data collection start date: July 2019 – end date: June 2020) are described in Table 1.\n\nPrognostic factors for knee pain in children and adolescents that can be measured in the context of general practice were identified from a review of the current literature on the topic31. Initially, the most important domains for the prognosis of knee pain and the specific items to be included in the tool were selected based on the strength of association from previous studies identified within the literature and from meta-analysis of individual participant data. Additional factors were included after discussion with researchers and clinicians with insight into this area (staff working at the Center of General Practice at Aalborg University). During this stage, great emphasis was given to include only the most necessary factors considering the ultimate use of the prognostic tool (i.e. it should be used in a reasonable time of a consultation within general practice). Items relative to the specific prognostic factors were initially selected from validated scales when possible (e.g. regarding pain characteristics, limitation in daily activities, sport participation, psychological factors, sleep), or from previous studies. When multiple items within a scale or multiple scales were available for a prognostic factor, the relevant literature on the topic was identified and discussed at meetings with GPs and staff working at the Center of General Practice at Aalborg University in order to select the most appropriate items within the possible options. Following previous tools developed for pain in children and adolescents19,29,32, items were properly worded for the age and properly framed with respect to the response options (e.g. direction, time intervals, avoiding double-barrelled items). A process of forward-backward translation of each item from Danish to English was applied to ensure that the items worded in Danish were conceptually equivalent to the items worded in English which were selected from previous studies or validated tools.\n\nThis project included a stage where the prognostic tool was piloted, which included a development, testing and implementation stage. After the initial development stage described above, the prognostic tool was tested with volunteer participants (n = 14) recruited through advertisement of the study on Facebook. Children and adolescents who were interested in the study (or their parents) contacted the primary investigator of this study (A.A.) for taking part in the test, and a date was arranged for testing the tool and carrying out cognitive interviews with a research assistant (T.S.). The test and cognitive interviews were carried out either in person at the Center for General Practice at Aalborg University or through Skype. The initial version of the tool was delivered to participants and asked to be completed, and the time needed for completion was assessed. After completion of the tool, cognitive interviews were carried out to assess the appropriateness, comprehensibility, wording and potential lack of items relating to the prognosis of knee pain, as previously done for other tools that evaluated pain status in children and adolescents29,32–34. The questionnaire for cognitive interviews is available as Extended data35. The aim was to improve the face, construct and content validity of the tool at this stage. This is especially important considering that a worse outcome can result if a general practitioner makes an inaccurate diagnosis of the child’s knee pain or if there is a lack of communication between the general practitioner and the child with knee pain36. After receiving feedback through cognitive interviews, the prognostic tool was implemented to reach the optimal final version to be used in the data collection stage (Figure 1; also available as Extended data27). Participants were given a cinema ticket as a reward for participating in the cognitive interviews.\n\nA pilot study to assess the stability of the prognostic tool (test-retest reliability33) in children and adolescents pre- and post- consultation was carried out. Children and adolescents who referred to primary care for knee pain and their parents were given the prognostic tool (together with the informed consent) to be completed in the waiting room of the general practice before the consultation. Subsequently, after the consultation with the general practitioner, children and their parents were asked to complete the prognostic tool again in order to assess the stability of the tool parameters and the general practitioner’s influence on the parameters (e.g. pain perception and psychological factors) assessed with the tool. Differences in reporting were assessed by means of K-statistics for categorical variables and intra-class correlation coefficient for continuous variable (e.g. age). Results, which are available as Extended data37, showed K-statistics values above 0.80 (range from 0.66 to 1) for most items, showing good stability. Only the item relative to helplessness had a value below 0.70 (K-statistics = 0.66), which is considered the minimum standard for reliability33.\n\nThe primary outcome measure will be the recurrence/persistence of activity-limiting knee pain (i.e. defined as participants reporting yes to having pain that is limiting activities in the same knee) at 3-month follow-up30. Participants will be asked about continuity of their knee pain if they report not having knee pain at follow-up (i.e. \"when did your knee pain stop?), to enable the distinction between recurrence (on/off knee pain episodes between baseline and follow-up) and persistence (continuous knee pain from baseline to follow-up) of knee pain.\n\nA secondary outcome measure that will be collected is the recurrence/persistence of activity-limiting knee pain at 6-month follow-up. In addition, previous studies have shown an effect of the treatment received on the change in risk group for the recurrence/persistence of pediatric pain38 and on the change in pain and function39. Therefore, an additional outcome measure that will be assessed include the treatment effectiveness on the recurrence/persistence of knee pain.\n\nA statistical analysis plan for the development of the final prognostic tool prior to recruitment has been published and can be publicly accessed40. The analysis plan includes the following stages:\n\n1. Descriptive analysis of the collected data, with results presented as means with SDs and as percentages.\n\n2. Assessment of potential floor and ceiling effects of the items included in the prognostic tool. This will be done by checking that for those items that represent an ordinal or categorical variable with more than two potential response categories, the responses given are not skewed towards the top or bottom extreme of the scales (e.g. a ceiling or floor effect is present if >15% of the respondents report the lowest/highest score of the scale30,33,41).\n\n3. Estimation of the knee pain prognosis (i.e. recurrence/persistence of knee pain, dichotomous outcome) at 3-month and 6-month follow-up by means of multiple logistic regression to estimate ORs and 95% confidence intervals for each item included in the tool. This will inform on which factors are most related to the prognosis of knee pain and will provide an insight on the scores of the prognostic tool (both overall and for subscales) to be applied for the creation of the initial risk groups. Alternatively, the RR will be estimated if another linear model analysis will be carried out.\n\n4. Discriminant validity of the prognostic score will be assessed by using receiver operating characteristics (ROC) curves and by calculating the area under the curve (AUC) for the overall score and subscales of the prognostic tool.\n\n5. Creation of risk groups for the recurrence/persistence of knee pain on the basis of cut-off scores identified using the ROC curves. The initial idea is to have two or three risk groups (e.g. low/medium/high), which have to be clinically meaningful. More importance will be given to the sensitivity of the tool over the specificity.\n\n6. Assessment of the predictive ability of the risk groups defined at baseline by calculating the sensitivity, specificity and negative and positive likelihood ratios (LRs) against the primary and secondary outcome (i.e. 3-month and 6-month recurrence/persistence of knee pain; disability/activities limitation due to knee pain).\n\n7. Assessment of the potential influence of non-modifiable patients’ characteristics on the predictive ability of the risk groups defined at baseline by stratifying the former analysis by age groups, sex and traumatic/non-traumatic onset.\n\nA sample size of minimum 300 participants from at least 20 general practices for the development of the tool has been estimated. This estimate was based on the following factors; the sample size required for the development of other prognostic tools19,20, the annual consultation prevalence for knee pain in children and adolescents in general practice, the size of general practices, the number of items in the tool and the rule of thumb of at least 10 events for variable (or items within the prognostic tool)33.\n\nPrevious studies have shown an annual consultation prevalence of 104–200 per 10,000 registered persons in children aged 3 to 19 years old42–44. Several potential scenarios about participants’ recruitment were hypothesized by considering the lowest and the highest annual consultation prevalence. These scenarios were calculated on a conservative estimate of 30% study participation rate. This is a worst-case scenario, and this approach was taken in order to have a safe recruitment that will provide enough children and adolescents for the development of the tool. These calculations resulted in an estimate of at least 20 general practices needed for recruiting the participants to the study (the estimate changes depending on the annual consultation prevalence considered and the size of the general practices; full calculations are available on request to the authors). In addition, if the recruitment from general practices will provide a low number of participants, a complementary recruitment through social media will be performed in order to achieve a total sample size of at least 300 children. In this case, sensitivity analysis would be performed to check for any potential difference in characteristics between the sample recruited through general practices and social media.\n\nThe participant submitted responses will be automatically registered in a database using the REDCap. Handling of data will comply to the General Data Protection Regulation and the concomitant local data handling instructions for Center of General Practice at Aalborg University. Data will be stored at a server at Aalborg University, this will ensure a safe and legal handling of data. The accuracy of the data will be checked through screening of data outliers and potentially “wrong” or “strange” data will be identified and corrected. In order to obtain a “full analysis set” for the project, participants will have to provide data from baseline through the 3-month and 6-month follow-up, which will allow to estimate the short-term and long-term knee pain prognosis. Data completeness (i.e. completion and accuracy of data forms) will be monitored and actions will be taken to overcome potential problems such as missing data45. In case of baseline missing data, the missing observation will be replaced by means of an imputation process (e.g. multiple imputation by chained equation) depending on the number of missing observations (i.e. multiple imputation is usually performed when the percentage of missing data is low). A sensitivity analysis will also be carried out in order to compare results between the analysis carried out on the dataset with missing observation (complete-case analysis) and the multiple imputed dataset (multiple imputation analysis). A backup copying of the dataset will be performed daily.\n\nThe final dataset will be accessed by A.A., M.S.R., M.B.J. and S.H..\n\nAny future protocol amendments or changes will be made publicly visible in the clinical trial registration, and clearly described in the subsequent reporting of the results.\n\nThe present study will provide data on the prognosis for knee pain in children and adolescents who present to primary care. In addition, this study will provide data on the usability of a prognostic tool to allocate children and adolescents to a category of risk for knee pain recurrence or persistence and consequently provide them with the best targeted treatment. The study results will be disseminated at scientific conferences and through appropriate scientific journals. General practitioners, children and parents participating into the study will be regularly provided with feedback about the ongoing study as well.\n\nAll authors of this current paper (AA, SH, MBJ, MSR) will be involved in the production of manuscripts originating from this study.\n\nEthic approval for the study and for the pilot study was seek by sending an enquiry to the Scientific Ethics Committee for Region North Jutland, together with a brief description of the study. The response obtained from the Scientific Ethics Committee stated that ethical approval was not needed, as the study implied the use of a questionnaire survey and did not imply any type of intervention on participants. Written informed consent will be obtained by the adolescents if aged 15 years old or more, otherwise from the parents. Participants who will become 15 years old during the transition from baseline to follow-up will be asked to provide consent themselves when contacted at follow-up.\n\n\nDiscussion\n\nThe objective of this study is to develop a user-friendly prognostic tool (Adolescent Knee Pain prognostic tool), which will be the first one to be used to support the GPs’ management of AKP. Within this study, the preliminary prognostic factors for AKP identified in the literature and included in the initial version of the tool will be tested. Those prognostic factors that will prove to be independently significantly associated with a poor AKP prognosis and will contribute to the prognostic model will be included in the final version of the tool. The tool will enable the identification of different subgroups of patients who seek primary care for AKP according to their risk of recurrence or persistence of knee pain at 3-month and 6-month follow-up.\n\nA limitation is that it might be argued that specific knee pain conditions (e.g. patellofemoral pain, Osgood-Schlatter) might be characterized by different prognostic courses. However, the accurate diagnosis of specific knee pain conditions in the primary care context might be challenging, and this tool was conceived for enquiring general questions regarding AKP. Second, this tool might not be applicable to health-care systems of countries where there might be a different categorization of primary care and secondary care or where GPs might not be the sole gatekeeper of primary care provision46. Third, there might be difficulties in recruiting 300 participants with AKP from general practices, and although alternative recruitment strategies have been planned (i.e. through social media), this might produce selection bias47.\n\nFirst, primary care is the place where the majority of health care is delivered48, and consequently the development of a tool to be used within this setting has the potential to have a significant impact in real-life.\n\nSecond, the prognostic tool includes items about factors that are specific to the AKP prognosis (e.g. pain duration, knee pain frequency) and therefore would be more sensitive than other more general pain tools. This is important considering that misclassification of patients might potentially lead to undertreat those misclassified as low-risk and overtreat those misclassified as high-risk49.\n\nThird, the subgroup of patients who refer to primary care is usually characterized by a different severity of symptoms compared to general population, second or tertiary care samples, as proposed by the iceberg theory of disease50–52. Hence, this research has the opportunity to provide information on the predictive ability of a prognostic tool in primary care compared to studies carried out within other care settings (e.g. the PPST was validated in tertiary care settings19,53), as it has previously been observed a difference in the efficacy of risk prediction potentially because of differences in patients case mix54.\n\nFourth, this prognostic tool is short (only 13 prognostic factors assessed overall) and quick to use (tests during the piloting of the tool showed that on average approximately three minutes and a half are needed to complete the tool) and includes factors that can be easily collected during a consultation with a GP. Finally, the tool is easy to be delivered and properly worded to be understandable by children and adolescents, as it was implemented following their feedback during cognitive interviews.\n\nThe use of this tool can potentially improve the understanding of the AKP prognosis and identify specific categories of risk of a poor prognosis. However, the care needed will differ among patients with AKP. Some of them will only need conservative management (e.g. education on how to manage knee pain, modification or avoidance of physical activity), while others will need a referral to a specialist (e.g. a physiotherapist, a rheumatologist). If it will prove to perform adequately (i.e. in terms of sensitivity, specificity, positive and negative likelihood ratio), this tool will inform on the likely prognosis of AKP and potentially guide the GPs in providing a targeted stratified care according to the risk of recurrence or persistence of AKP at 3-month and 6-month follow-up. The use of this tool can potentially significantly change the use of resources and increase the primary care efficiency by allocating resources to those who need them most.\n\n\nTrial status\n\nName of registry: ClinicalTrials.gov\n\nRegistration number: NCT03995771\n\nDate of registration: 24/06/2019\n\nDate of start of recruitment: 01/07/2019\n\nExpected date of recruitment conclusion: 30/06/2020\n\nTrial URL: https://clinicaltrials.gov/ct2/show/NCT03995771?term=NCT03995771&rank=1.\n\n\nData availability\n\nNo underlying data are associated with this article.\n\nHarvard Dataverse: Dataset to test the stability of the prognostic tool. https://doi.org/10.7910/DVN/SMQRKA37.\n\nThis project contains the dataset generated when testing the stability of the tool.\n\nHarvard Dataverse: Questionnaire for cognitive interviews. https://doi.org/10.7910/DVN/UT1MGD35.\n\nThis project contains the Danish- and English-language questionnaire for cognitive interviews.\n\nHarvard Dataverse: Final questionnaire. https://doi.org/10.7910/DVN/QKWOOT27.\n\nThis project contains the final Danish- and English-language questionnaire for data collection.\n\nHarvard Dataverse: SPIRIT checklist for ‘The Adolescent Knee Pain (AK-Pain) prognostic tool: protocol for a prospective cohort study’. https://doi.org/10.7910/DVN/SIO5WG21.\n\nExtended data and completed reporting guidelines checklist are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Author contributions\n\n\n\nAA planned the overall study protocol and all other authors (SH, MBJ and MSR) provided clinical advice and contributed to the refinement of the protocol. AA led the writing of this study protocol paper, and all other authors (SH, MBJ and MSR) contributed equally with comments and critical revision to the manuscript. All authors read and approved the final manuscript.\n\n\nAcknowledgments\n\nWe would like to thank all the GPs and their staff who agreed to take part to the project and research assistant Tagrid Salim (T.S.) for carrying out the cognitive interviews. We would also like to thank all the children and adolescents (and their families) who took part in the piloting of the prognostic tool and provided valuable advice for the improvement of the tool.\n\n\nReferences\n\nRathleff MS, Rathleff CR, Olesen JL, et al.: Is Knee Pain During Adolescence a Self-limiting Condition? Prognosis of Patellofemoral Pain and Other Types of Knee Pain. Am J Sports Med. 2016; 44(5): 1165–71. PubMed Abstract \| Publisher Full Text\n\nHuguet A, Tougas ME, Hayden J, et al.: Systematic review with meta-analysis of childhood and adolescent risk and prognostic factors for musculoskeletal pain. Pain. 2016; 157(12): 2640–56. PubMed Abstract \| Publisher Full Text\n\nGroenewald CB, Essner BS, Wright D, et al.: The economic costs of chronic pain among a cohort of treatment-seeking adolescents in the United States. J Pain. 2014; 15(9): 925–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHenschke N, Harrison C, McKay D, et al.: Musculoskeletal conditions in children and adolescents managed in Australian primary care. BMC Musculoskelet Disord. 2014; 15: 164. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRathleff MS, Holden S, Straszek CL, et al.: Five-year prognosis and impact of adolescent knee pain: a prospective population-based cohort study of 504 adolescents in Denmark. BMJ Open. 2019; 9(5): e024113. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRathleff CR, Olesen JL, Roos EM, et al.: Half of 12-15-year-olds with knee pain still have pain after one year. Dan Med J. 2013; 60(11): A4725. PubMed Abstract\n\nRathleff MS: Patellofemoral pain during adolescence: much more prevalent than appreciated. Br J Sports Med. 2016; 50(14): 831–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaraldstad K, Sørum R, Eide H, et al.: Pain in children and adolescents: prevalence, impact on daily life, and parents' perception, a school survey. Scand J Caring Sci. 2011; 25(1): 27–36. PubMed Abstract \| Publisher Full Text\n\nKosola S, Mundy LK, Sawyer SM, et al.: Pain and learning in primary school: a population-based study. Pain. 2017; 158(9): 1825–30. PubMed Abstract \| Publisher Full Text\n\nSandow JM, Goodfwllow JW: The natural history of anterior knee pain in adolescents. J Bone Joint Surg Br. 1985; 67(1): 36–8. PubMed Abstract\n\nHolm S, Ljungman G, Söderlund A: Pain in children and adolescents in primary care; chronic and recurrent pain is common. Acta Paediatr. 2012; 101(12): 1246–52. PubMed Abstract \| Publisher Full Text\n\nBuck R, Wynne-Jones G, Varnava A, et al.: Working with Musculoskeletal Pain. Rev pain. 2009; 3(1): 6–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeat G, McCarney R, Croft P: Knee pain and osteoarthritis in older adults: a review of community burden and current use of primary health care. Ann Rheum Dis. 2001; 60(2): 91–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJinks C, Jordan K, Ong BN, et al.: A brief screening tool for knee pain in primary care (KNEST). 2. Results from a survey in the general population aged 50 and over. Rheumatology (Oxford). 2004; 43(1): 55–61. PubMed Abstract \| Publisher Full Text\n\nThomas E, Dunn KM, Mallen C, et al.: A prognostic approach to defining chronic pain: application to knee pain in older adults. Pain. 2009; 139(2): 389–97. PubMed Abstract \| Publisher Full Text\n\nHestbaek L, Leboeuf-Yde C, Kyvik KO: Are lifestyle-factors in adolescence predictors for adult low back pain? A cross-sectional and prospective study of young twins. BMC Musculoskelet Disord. 2006; 7: 27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeat G, Riley RD, Croft P, et al.: Improving the transparency of prognosis research: the role of reporting, data sharing, registration, and protocols. PLoS Med. 2014; 11(7): e1001671. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCroft P, Altman DG, Deeks JJ, et al.: The science of clinical practice: disease diagnosis or patient prognosis? Evidence about \"what is likely to happen\" should shape clinical practice. BMC Med. 2015; 13(1): 20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSimons LE, Smith A, Ibagon C, et al.: Pediatric Pain Screening Tool: rapid identification of risk in youth with pain complaints. Pain. 2015; 156(8): 1511–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHill JC, Dunn KM, Lewis M, et al.: A primary care back pain screening tool: identifying patient subgroups for initial treatment. Arthritis Rheum. 2008; 59(5): 632–41. PubMed Abstract \| Publisher Full Text\n\nAndreucci A: \"SPIRIT checklist\". Harvard Dataverse, V1. 2019. http://www.doi.org/10.7910/DVN/SIO5WG\n\nRiis A, Jensen CE, Maindal HT, et al.: Recruitment of general practices: Is a standardised approach helpful in the involvement of healthcare professionals in research? SAGE Open Med. 2016; 4: 2050312116662802. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvon Baeyer CL, Lin V, Seidman LC, et al.: Pain charts (body maps or manikins) in assessment of the location of pediatric pain. Pain Manag. 2011; 1(1): 61–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMichaleff ZA, Kamper SJ, Stinson JN, et al.: Measuring Musculoskeletal Pain in Infants, Children, and Adolescents. J Orthop Sport Phys Ther. 2017; 47(10): 712–30. PubMed Abstract \| Publisher Full Text\n\nStinson JN, Kavanagh T, Yamada J, et al.: Systematic review of the psychometric properties, interpretability and feasibility of self-report pain intensity measures for use in clinical trials in children and adolescents. Pain. 2006; 125(1–2): 143–57. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: WHO - Definition of key terms. 2013; [cited 2018 Sep 3]. Reference Source\n\nAndreucci A: \"Final questionnaire\". Harvard Dataverse, V1. 2019. http://www.doi.org/10.7910/DVN/QKWOOT\n\nHarris PA, Taylor R, Thielke R, et al.: Research electronic data capture (REDCap)--a metadata-driven methodology and workflow process for providing translational research informatics support. J Biomed Inf. 2009; 42(2): 377–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nÖrtqvist M, Roos EM, Broström EW, et al.: Development of the Knee Injury and Osteoarthritis Outcome Score for children (KOOS-Child): comprehensibility and content validity. Acta Orthop. 2012; 83(6): 666–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrtqvist M, Iversen MD, Janarv PM, et al.: Psychometric properties of the Knee injury and Osteoarthritis Outcome Score for Children (KOOS-Child) in children with knee disorders. Br J Sports Med. 2014; 48(19): 1437–46. PubMed Abstract \| Publisher Full Text\n\nPourbordbari N, Riis A, Jensen MB, et al.: Poor prognosis of child and adolescent musculoskeletal pain: a systematic literature review. BMJ Open. 2019; 9(7): e024921. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIversen MD, Lee B, Connell P, et al.: Validity and comprehensibility of the International Knee Documentation Committee Subjective Knee Evaluation form in Children. Scand J Med Sci Sport. 2010; 20(1): e87–95. PubMed Abstract \| Publisher Full Text\n\nTerwee CB, Bot SD, de Boer MR, et al.: Quality criteria were proposed for measurement properties of health status questionnaires. J Clin Epidemiol. 2007; 60(1): 34–42. PubMed Abstract \| Publisher Full Text\n\nVarni JW, Stucky BD, Thissen D, et al.: PROMIS Pediatric Pain Interference Scale: An Item Response Theory Analysis of the Pediatric Pain Item Bank. J Pain. 2010; 11(11): 1109–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndreucci A: \"Questionnaire for cognitive interviews\". Harvard Dataverse, V1. 2019. http://www.doi.org/10.7910/DVN/UT1MGD\n\nRobins H, Perron V, Heathcote L, et al.: Pain Neuroscience Education: State of the Art and Application in Pediatrics. Children (Basel). 2016; 3(4): pii: E43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndreucci A: \"Final dataset without identifiers.tab\", Dataset to test the stability of the prognostic tool. 2019; Harvard Dataverse, V1. http://www.doi.org/10.7910/DVN/SMQRKA/6IUDQG\n\nVuorimaa H, Leppänen L, Kautiainen H, et al.: Risk severity moderated effectiveness of pain treatment in adolescents. Scand J Pain. 2019; 19(2): 287–98. PubMed Abstract \| Publisher Full Text\n\nPalermo TM, Law EF, Zhou C, et al.: Trajectories of Change During a Randomized Controlled Trial of Internet-delivered Psychological Treatment for Adolescent Chronic Pain: How does Change in Pain and Function Relate? Pain. 2015; 156(4): 626–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClinicalTrials.gov: Adolescent Knee Pain (AK-Pain) Prognostic Tool - Study Record Detail. [cited 2019 Jul 6]. 2019. Reference Source\n\nKocher MS, Smith JT, Iversen MD, et al.: Reliability, validity, and responsiveness of a modified international knee documentation committee subjective knee form (Pedi-IKDC) in children with knee disorders. Am J Sports Med. 2011; 39(5): 933–9. PubMed Abstract \| Publisher Full Text\n\nMichaleff ZA, Campbell P, Protheroe J, et al.: Consultation patterns of children and adolescents with knee pain in UK general practice: analysis of medical records. BMC Musculoskelet Disord. 2017; 18(1): 239. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Inocencio J: Musculoskeletal pain in primary pediatric care: analysis of 1000 consecutive general pediatric clinic visits. Pediatrics. 1998; 102(6): E63. PubMed Abstract \| Publisher Full Text\n\nTan A, Strauss VY, Protheroe J, et al.: Epidemiology of paediatric presentations with musculoskeletal problems in primary care. BMC Musculoskelet Disord. 2018; 19(1): 40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRiis A, Jensen CE, Bro F, et al.: Enhanced implementation of low back pain guidelines in general practice: study protocol of a cluster randomised controlled trial. Implement Sci. 2013; 8: 124. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKringos D, Boerma W, Bourgueil Y, et al.: The strength of primary care in Europe: An international comparative study. Br J Gen Pract. 2013; 63(616): e742–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDelgado-Rodríguez M, Llorca J: Bias. J Epidemiol Community Health. 2004; 58(8): 635–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCardy A, Holden S, Watson D, et al.: Recruiting children onto research studies by the Scottish Primary Care Research Network: A real team effort. Qual Prim Care. 2012; 20(3): 199–206. PubMed Abstract\n\nKarran EL, McAuley JH, Traeger AC, et al.: Can screening instruments accurately determine poor outcome risk in adults with recent onset low back pain? A systematic review and meta-analysis. BMC Med. 2017; 15(1): 13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCampbell SM, Roland MO: Why do people consult the doctor? Fam Pract. 1996; 13(1): 75–83. PubMed Abstract \| Publisher Full Text\n\nLast JM, Adelaide DP: The iceberg: 'completing the clinical picture' in general practice. 1963. Int J Epidemiol. 2013; 42(6): 1608–13. PubMed Abstract \| Publisher Full Text\n\nBhopal R: Concepts of Epidemiology - an integrated introduction to the ideas, theories, principles and methods of epidemiology. 2nd ed. Oxford University Press. 2002; 456. Reference Source\n\nHeathcote LC, Rabner J, Lebel A, et al.: Rapid screening of risk in pediatric headache: Application of the pediatric pain screening tool. J Pediatr Psychol. 2018; 43(3): 243–51. PubMed Abstract \| Publisher Full Text\n\nMorsø L, Kent P, Manniche C, et al.: The predictive ability of the STarT Back Screening Tool in a Danish secondary care setting. Eur Spine J. 2014; 23(1): 120–8. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "58102", "date": "27 Jan 2020", "name": "Signe Fuglkjær Møller", "expertise": [ "Reviewer Expertise musculoskeletal health" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for giving me the opportunity to review this well described paper about development and test of a prognostic tool for knee pain in adolescence. A very important area. I am very pleased to see the incorporation of psykosocial aspect in the pain.\nI was wondering about the use of the word Ængstelig/deprimeret (anxious/depressed). Do children aged 8 and a bit older understand that word? Would it be better to use the word bekymret (worried) instead?\n\nIn the statistics section; you write that you will stratify by traumatic/non-traumatic onset. Why do they choose to do so?\n\nI think this paper is well written and described and I therefore have no further comments. I am looking forward the seeing the results.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "5238", "date": "21 Feb 2020", "name": "Alessandro Andreucci", "role": "Author Response", "response": "Dear Editorial Team, F1000 Research, We were delighted that the Reviewers of our recent submission \" The Adolescent Knee Pain (AK-Pain) prognostic tool: protocol for a prospective cohort study\" (ID: 21740) provided us with their very helpful feedback on our paper. This has given us the opportunity to apply changes that have led to an improvement of the paper. Reviewer 1 Many thanks for the opportunity to review this protocol paper that reports on the rationale and methodology of testing an adolescent knee pain prognostic tool. The protocol is generally well written, and the proposed project to develop a prognostic tool is much needed within the child and adolescent musculoskeletal community. However I do feel some points within the paper require greater clarity and have set out a number of recommendations that I feel would improve the content and presentation of this paper. Response: We thank the reviewer for the valuable feedback provided. We have addressed the comments and changed the manuscript (changes are underlined in the text), which we feel is now improved. Individual responses for each specific comment are outlined below.1. Whilst the authors have aptly discussed the rationale for a prognostic tool within the introduction and briefly touch upon stratified care, I feel they need also mention what this means in terms of aligned treatments based on the risk (i.e. the pathway for stratified patients), a little bit more on this would benefit the introduction.Response: We thank the reviewer for the comment and have now amended the introduction by providing more information on the aligned treatments based on the risk.The text (Introduction section) now reads “One potential option to support clinical decision making is to develop decision aids such as prognostic tools. These tools often consist of items with prognostic value that can be asked during the clinical consultation or completed prior to the consultation. Thereby patients can be stratified depending on their prognosis (19) and subsequently the best targeted treatment based on the prognostic profile can be offered the patient. In the case of a child or adolescent with knee pain, different treatments or recommendations (e.g. short education session, modification of physical activity levels, exercise, use of painkillers, referral to a specialist) might be provided depending on the risk of a poor prognosis. Examples of prognostic tools that have already been developed include the Keele STarT Back Screening Tool (SBST) for lower back pain in adults (20) or the Pediatric Pain Screening Tool (PPST) for general pediatric pain (19).”2. In the exclusion criteria the authors mention participants will be excluded if they have an inability to understand the questionnaire, is this a language issue, or for those with learning difficulties, or both, I think the authors might wish to expand on this a little more to give examples.Response: Participants are meant to be excluded if they have either language issues or learning difficulties.The text (section Methods, Eligibility and recruitment of participants) now reads “Inability to take part in the study due to an inability to understand the questionnaire (i.e. participants who have issues with Danish language or have learning difficulties)” 3. The authors state that both parents will be approached before the consultation, does that then require that both parents be present as this won’t often be the case (parent working, single parent etc), also more fundamental is the issue of “before the consultation”, if this is the case how do they know that the knee pain is not referred pain from the spine for example or that whilst the child/adolescent has knee pain that they may also discuss other issues within the consultation and the focus is on those other issues? I might add that the child may be looked after by someone who is not the parent (family member) and perhaps the term “caregiver” should also be used when the term parent is used?Response: In Denmark, the general practitioner and his/her staff know in advance if the person is consulting for knee pain based on the booking. However, the general practitioner might also be made aware of the knee pain problem during a consultation regarding other issues. In this case, the questionnaire will be filled out after the consultation. As the reviewer correctly pointed out however, the knee pain might be referred from other body sites (e.g. spine), and we acknowledge that this is a limitation of our study. We have amended the text and made it clearer. The text (section Methods - Eligibility and recruitment of participants) now reads “Participants will be recruited from general practices on the basis of consultation for knee pain. In Denmark, consultations with the GP are booked beforehand, which allows the GP and the GP’s staff to know in advance what the patient is consulting for. The study will be introduced to the caregivers and children before the consultation by a person working at the general practice (e.g. the GP/nurse/secretary). If patients meet the inclusion criteria for being eligible into the study (e.g. patient aged between 8 and 19 years old who consult for knee pain) and agree to participate, they will be provided with the study material (i.e. prognostic tool to be completed). However, if the GP was made aware of the issue of the presence of knee pain during a consultation regarding other health issues, the questionnaire will be completed after the consultation.” 4. The authors describe complementary recruitment strategies to work alongside primary care recruitment, I think the use of wider recruitment strategy is good considering the low participation rate of children within primary care to MSK studies. However, whilst the authors have stipulated that children/adolescents will be reminded of the premise for participation, i.e. that participants have been seen by their GP for knee pain, how will this be confirmed? Furthermore, the authors have not explained the timeline for this, for example the recruitment within GP practices is at the time of consultation, however social media recruitment does not state a criteria of when they should have consulted. One of the key prognostic markers for adult MSK pain is time since onset, here you may have potentially two distinct groups, one who have consulted (not clear whether this will be the first consultation or a subsequent visit), and those via social media at presumably post consultation (but not stipulated as to what time period criteria is acceptable, is it a week, month, year etc), this presents issues of case mix, and at the very least the authors would have to collect information about timing of consultations (and timing of when patient first experienced pain) to factor into their analysis.Response: We thank the reviewer for the very insightful comment.Our plan is to include only those who have consulted within one week. In addition, sensitivity analysis will be applied by comparing results of those recruited in primary care and those recruited through social media to assess potential differences related to case mix. Regarding timing of when patient first experienced pain, this is already assessed with item number 4 of the prognostic tool. We have amended the text and made it clearer. The text (section Methods - Eligibility and recruitment of participants) now reads “Complementary recruitment strategies that might be applied to maximize the recruitment rate are the advertisement of the study through social media (e.g. Facebook, Twitter, Snapchat) and through explicative posters displayed at the participating general practices. In this case, it will be screened that participants have been seen by their GP within the last week and knee pain was part of the consultation.”5. I think the offer of cinema tickets is a good incentive, however would it not be better to offer a family ticket or at least two tickets as a child would most likely go on their own?Response: Although the idea of offering a family ticket or at least two tickets is good, unfortunately we were limited in our choice by financial constrictions. This is why we can offer only one cinema ticket at baseline and one cinema ticket at follow-up, and unfortunately it is not possible to change this strategy now as the study is already on-going.6. For the data collection section, I wonder if a diagram might be helpful here as it is not fully clear, does the GP collect the data post consultation, will they (GPs) have time to do this, the authors mention previously that the child/adolescent will be approached to complete the tool before consultation, so does this mean they fill in the tool before consultation and then other information post consultation? The authors should be aware that pre-consultation may reveal different responses to a post-consultation (e.g. reductions in fear avoidance for parent/child due to reassurance from GP etc), this point also relates to point 4 above where alternative recruitment strategies may be used that could lead to different responses due to different timings?Response: We thank the reviewer for the very insightful comment.Data collection will occur before the consultation and will be facilitated by the general practitioner’s staff (e.g. nurse, secretary), with the tool to be fully completed before consultation. However, as pointed out at point 3 above, the knee pain problem might not be cited as the primary reason for consultation, and the GP only made aware of it during the consultation. In this case, the tool will be completed after the consultation. Being aware of the potential differences in parameters pre-consultation vs. post-consultation we assessed the stability of the tool, which showed good stability of the tool parameters before and after consultation with the general practitioner, as described in section “Methods – Stability of the tool”. 7. When discussing the outcomes, the authors describe that outcomes will be collected by a combination of self-report or parent report, firstly this doesn’t make sense because of the inclusion of “or” as that implies either, whereas it is described as a combination. Does this mean that there will be a choice in who fills in the questionnaire, should it not be self-report AND parent report, or is this related to age and access to online social media platforms where parents/caregivers would have to complete, I think this needs to be explained, and again the researchers should collect information on who fills in the questionnaire as there are differences between proxy and self-report.Response: We thank the reviewer for the comment. The outcome will be collected by questionnaires that will be sent through an e-mail with a link to the web application for online surveys or text message. Within the questionnaire, there are also questions about name, surname and contact information (e-mail and phone number) and about who the person replying to the questionnaire is (i.e. child alone/caregiver/child together with the caregiver). This allows us to know if the person replying is the caregiver or the child/adolescent.We have amended the text and made it clearer. The text (section Methods – Data collection) now reads: “Outcomes will be collected by questionnaires. The adolescent can choose to do this self-reported or caregiver-reported (through an e-mail with a link to the web application for online surveys or text message) at follow-up (two time points: 3-month and 6-month follow-up). The questionnaire also includes a question about who the person replying to the questionnaire is (i.e. child alone/caregiver/child together with the caregiver)” 8. Participant timeline, is table 1 needed, I think it is clear that there will be an assessment at baseline and follow up at 3 months and 6 months for each participant, I don't think a diagram is needed for that? I feel a recruitment flow diagram would be a much better inclusion for this paper.Response: We have replaced table 1 with a recruitment flow diagram and modified the text, which now reads “The process of recruitment and retainment of participants in the study (data collection start date: July 2019 – end date: June 2020) is described in the following flow diagram (figure 1).” 9. The authors discuss the development of the items for the tool, I think a little more should be said about this, for example is this the information within the review (ref 31) or additional literature, also as this is a generation of the candidate items for a prognostic tool it would be good to give some detail (e.g. how many studies within the review, what sort of populations, were they all prospective studies etc). In addition the authors discuss the situation where multiple items or multiple scales were present for items, I feel this whole sentence could do with expansion, what is meant by multiple items and multiple scales, do you mean prognostic factors that have been measured suing multiple items/scales? Give some examples and how a multiple scale construct is then reduced to a single question, how was this done?Response: We thank the reviewer for the insightful comment.Items to be included in the prognostic tool were selected based on a combination of those initially identified with the review (31), of the wider literature on child and adolescent musculoskeletal pain and from meta-analysis of individual participant data (PROSPERO ID CRD42019116861). This allowed us to include also other potential factors that were not previously assessed in the studies identified in the review but were found to be associated with the prognosis of general musculoskeletal pain in other studies and might therefore result as significant contributors to the prognostic model. Regarding the situation where multiple items or multiple scales were present for items relative to the prognostic factors, single items were selected from the most appropriate scales. We have now modified the text (Section Methods - Development of the tool) and made it clearer. The text now reads: “Prognostic factors for knee pain in children and adolescents that can be measured in the context of general practice were identified from a review of the current literature on the topic (29). The review included 26 prospective studies, of which 4 focused on knee pain. These 4 studies included schoolchildren 12-19 years old from Denmark (3 studies) and 10-12 years old from Finland. Initially, the most important domains for the prognosis of knee pain and the specific items to be included in the tool were selected based on the review but also from strength of association from previous studies identified within the literature and from meta-analysis of individual participant data. Prognostic factors for knee pain identified within the review (29) were increasing age, pain frequency, practicing sport more than 2 times/week and low quality of life (second question of item 9 within the tool). Other factors that were identified from the wider literature and meta-analysis of individual participant data (PROSPERO ID CRD42019116861) were knee pain characteristics (pain duration, traumatic/non-traumatic pain onset, limitations in daily activities due to knee pain, presence of knee pain in one knee or both knees), presence of pain in other body sites, gender, sleep, smoking, psychological factors, parental history of pain. During this stage, great emphasis was given to include only the most necessary factors considering the ultimate use of the prognostic tool (i.e. it should be used in a reasonable time of a consultation within general practice). Items relative to the specific prognostic factors were initially selected from validated scales when possible (e.g. regarding pain characteristics, limitation in daily activities, sport participation, psychological factors, sleep), or from previous studies. When multiple items within a scale or multiple scales were available for a prognostic factor, the relevant literature on the topic was identified and discussed at meetings with GPs and staff working at the Center of General Practice at Aalborg University in order to select the most appropriate items within the possible options. For example, the sleep item was selected from the self-reported Pediatric Quality of Life Inventory, one question of the psychological factor item from the Pain Catastrophizing Scale for Children, one question of the psychological factor item and the item relative to limitations in daily activities from the EuroQol Five-Dimensional Questionnaire Youth version.” 10. Within the study piloting section, the authors mention the process of improving face, construct, content validity, stressing the importance of this should a GP make an inaccurate diagnosis, I have some concern about this statement as it suggests that the questions asked about pain via the tool are in some way involved in diagnosis, this is surely not the case, can the authors explain what they mean?Response: The aim of the study piloting was to improve the comprehensibility and wording of the questions included in the tool, so that adolescents would provide information about the parameters related to the knee pain prognosis that were as close as possible to the real values and therefore limit potential misreporting. As the reviewer correctly pointed out, the tool is not conceived as a replacement of the diagnosis made by the general practitioner, but only as a support for understanding the prognosis together with the clinical examination. We have now modified the text and made it clearer. The text (section Methods – Study piloting) now reads: “The aim was to improve the face, construct and content validity of the tool at this stage. This is especially important considering that a worse outcome can result if there is a lack of communication between the GP and the child about the knee pain characteristics and the factors related to knee pain assessed within the tool (33).”11. In the stability of the tool section the authors state that children were referred to primary care because of their knee pain, who is it then who refers children to primary care, what is the process for this as this may be a factor to be considered (e.g. how long does it take for a person to be referred to primary care, who does the referral, how are decisions made on a referral etc), I thought it was that the child/parent/caregiver attends primary care?Response: We have amended the imprecision in the text, as we actually meant the child/adolescent who consult primary care for knee pain. We have modified the text, which now reads “Children and adolescents who consulted primary care and their caregivers were given the prognostic tool (together with the informed consent) to be completed in the waiting room of the general practice before the consultation.”12. I am slightly confused about the test-retest parameters, from what is written it appears that the test was done pre and post consultation (i.e. within the same day, perhaps within the same hour), this timeline is too short, the point of test/retest is to ensure that the participants would not remember the responses to the questions asked, so for example the next week, to then ensure that the responses are consistent, I am not convinced this current test stacks up, and any differences would likely be via information received within the consultation (influencing different responses) rather than the actual difference coming from inconsistency in the measure (for example a GP offering reassuring information).Response: We thank the reviewer for the comment and acknowledge the inaccuracy in the language used within the text. As pointed out by the reviewer, the GP might influence the perception of certain parameters related to pain (i.e. psychological factors, pain duration) through the discussion occurring during the consultation. In order to test if responses to the questions asked were influenced by the consultation with the GP (and to estimate the difference in responses), the stability of the tool before and after the consultation was tested and results showed good stability. However, as the reviewer correctly pointed out, this cannot be considered a test-retest as the time frame between the two measurements was too short. We have amended the text, (Section Methods – Stability of the tool) which now reads “A pilot study to assess the stability of the prognostic tool (31) in children and adolescents pre- and post- consultation was carried out.”13. I feel for the general readership it would be helpful to have the tool in English within the paper, and when I tracked down the English version I could see it has 12 questions, whereas the one in the current paper has 13 questions?Response: The English version, with 13 questions is accessible at: https://doi.org/10.7910/DVN/QKWOOT 14. The secondary outcome is the same as the primary outcome, with the only difference being time, could the authors clarify whether this is actually a secondary outcome or just a different time period for the same primary outcome measure?Response: We thank the reviewer for the comment and have amended the imprecision in the text. In our study, the outcome is the same is collected at two endpoints. We decided to consider the knee pain prognosis at 3-month as the primary endpoint and the knee pain prognosis at 6 months as a secondary endpoint.We have modified the text (Section Methods – Outcome), which now reads “The outcome measure will be the recurrence/persistence of activity-limiting knee pain (i.e. defined as participants reporting yes to having pain that is limiting activities in the same knee) at follow-up (28). Participants will be asked about continuity of their knee pain (i.e. “do you still have knee pain?” and, if they reply “no”, “when did your stop having knee pain?”), to enable the distinction between recurrence (on/off knee pain episodes between baseline and follow-up) and persistence (continuous knee pain from baseline to follow-up) of knee pain. The primary end-point that will be collected is the recurrence/persistence of activity-limiting knee pain at 3-month follow-up, while the secondary end-point is the recurrence/persistence of activity-limiting knee pain at 6-month follow-up” 15. The authors state that the statistical analysis plan can be found elsewhere, this protocol paper should include the full statistical analysis plan rather than have a signpost to another protocol, can the authors include the full details in this paper (and if they have then they don’t need to reference another protocol).Response: We have now corrected the text (section Methods – Statistics), inserted the full analysis plan within this paper and deleted the reference to the other protocol. 16. The authors discuss potential floor and ceiling effects, can the authors say a little more about this as presumably some risk items would only identify those at extremes (i.e. at risk) in any case? This is particularly true of items that can identify those at high risk, they may only account for a small proportion of the population with a number of items giving an additive high risk score?Response: We thank the reviewer for the very insightful comment. As correctly pointed out by the reviewer, for items that represent an ordinal or categorical variable with more than two potential response categories (for example 5 categories), the responses given might be only the top or bottom extreme of the scales. For example, this might happen for items related to psychological factors and sleep. In this case, participants with higher levels of anxiety/depression, helplessness or sleep problems would most likely fall in the high-risk category. A potential option to account for this is to initially test the independent effect of each item by means of multiple logistic regression, include those items that show a significant contribution to the model and subsequently apply different weights based on the strength of association. For example, a combination of the different items can be used to identify the scores for defining the risk groups (i.e. low/medium/high) and if items that identify only a small proportion of the population (e.g. high levels of psychological factors or sleep problems) are associated with a high risk of a bad prognosis, they can be selected and used to give an additive high-risk score. 17. Will the authors examine both each individual item but also within a model (multivariable) to test the independent effects of each item as some many have overlapping qualities (will they also examine redundant items); also will the authors consider model fit (e.g. explained variance) within their analysis?Response: We thank the reviewer for the comment. The plan is to test the independent effect of each item by means of multiple logistic regression and include only those items that show a significant contribution to the model. Therefore, the items that will not show a significant contribution will be excluded. In addition, items might be weighted differently based on the strength of association. We have modified the text (section Methods – Statistics), which now reads: “We will estimate the knee pain prognosis(i.e. recurrence/persistence of knee pain, dichotomous outcome) at 3-month and 6-month follow-up by means of multiple logistic regression to estimate ORs and 95% confidence intervals for each item included in the tool. This allows to assess the independent effects of each item and will inform on which factors are most related to the prognosis of knee pain (only the items that will show a statistically significant contribution to the model will be selected). This will also provide an insight on the scores of the prognostic tool (both overall and for subscales) to be applied for the creation of the initial risk groups. Alternatively, the RR will be estimated if another linear model analysis will be carried out. In addition, a potential option is to apply different weights to the items based on the strength of association.” 18. Point 5 in the statistical plan is not explained well, could the authors say a little more about the identification of cut off scores and what criteria will be used to decide on this (i.e. are these independently generated from other sample or this one, what determines a poor outcome, if determined from this cohort it will make external validity more problematic), also if more will be given to sensitivity of the tool, how will this factor in the development of risk groups? Also it might be beneficial for the authors to compare risk groups in terms of the baseline characteristics and test across those to give further discriminant information, and in addition it might be worth considering construct validity by comparing to any existing measures, for example this measure could be compared to the generic PPST?Response: We thank the reviewer for the insightful comment and provide some clarifications on this point.At each follow-up time-point, a poor outcome is defined as the presence of activity-limiting knee pain. As the cut-off scores will be generated by using data of this sample, the prognostic model developed within this cohort will have to be externally validated with another cohort in the future. Regarding sensitivity, we stated that more importance will be given to sensitivity of the tool, meaning that we will choose cut-off scores for the definition of risk groups that will allow to identify the majority of those with a potential bad prognosis in the high risk group, and rule out from the group those with a good prognosis. In addition, despite comparing the prognostic tool to the pediatric pain screening tool might be an option, it should be taken into account that the pediatric pain screening tool was validated in a tertiary care clinic and may operate differently in a primary care setting, which is where our prognostic tool is meant to operate. Therefore, the comparison might result in a poor match between risk group categories of the two tools due to differences in the severity of the sample used to develop the tools. We have modified the text, which now reads: “Data from this sample will be used for the creation of risk groups for the recurrence/persistence of knee pain, on the basis of cut-off scores identified using the ROC curves. Weights based on the strength of association identified with the multiple logistic regression might be applied. The initial idea is to have two or three risk groups (e.g. low/medium/high), which have to be clinically meaningful. More importance will be given to the sensitivity of the tool over the specificity. This means that in the presence of different cut-off scores for the inclusion of patients in the high-risk group, the cut-off that will allow to identify the majority of those with a bad prognostic outcome will be chosen. This will be done to avoid the misclassification of patients at high-risk in the medium or low-risk group.” 19. The discussion might benefit more by describing the potential for the tool in terms of the assistance to clinicians, for example within the study will the research consider how clinicians feel about the tool and whether they feel it would be useful, also what is not clear at present is the potential pathways that are available as defined by risk, do the proposed risk groups have an aligned pathway already for example, if so what might that look like and if not how will these be developed?Response: We thank the reviewer for the insightful comment and provide some clarifications on this point.There are other steps that will follow the development of this tool. As the reviewer highlights, there are additional steps to be completed before implementing the tool. This includes 1) an external validation of the tool 2) developing the matched care pathways 3) mixed-methods studies to understand how the tool should look and operate in the context of general practice, and 4) subsequent clinical trials to test the impact of the tool. A cluster randomized controlled trial will be performed where patients will be randomized to stratified care provided by using the tool or to the best current care, and differences in clinical outcomes will be assessed. The original idea is to provide targeted treatments to the three risk groups. For example, patients in the low risk group might be provided with an initial short patient education session and given a leaflet that reinforces the information given by the GP. For the medium risk group, strengthening exercises and load management might be delivered, while for the high-risk group a tailored treatment might be delivered that will target also psychological factors. However, these are only initial suggested treatments, and additional treatments might be developed depending on the items that will prove to be significantly associated with a bad prognosis (e.g. potential further treatments might be advices regarding having a good sleep hygiene). Regarding assistance to the clinician, this tool together with another tool for the knee pain diagnosis, will be part of a package (decision tool) that will assist clinicians in the diagnosis and treatment of knee pain. Future qualitative studies will be needed to assess the GPs´ behaviour when using the tool. We have now modified the text and added this part to the section \"Use of the tool for providing stratified care\":“The development of this tool fits within a wider research program, which overall aim is to provide a stratified approach to primary care management of child and adolescent knee pain that can result in clinical and economic benefits compared with current best practice. Therefore, future perspectives include the use of this tool in a randomized controlled trial, which will investigate whether subgrouping patients using the tool, combined with targeted treatment, is more clinically effective (i.e. it will reduce long- term disability from knee pain) and cost-effective compared to best current care. In addition, there is scope for performing future qualitative studies to assess the GPs´ behavioral change when using the tool (e.g. changes in referral to physical therapy, diagnostic tests and medication prescriptions)” Reviewer 2 Thank you for giving me the opportunity to review this well described paper about development and test of a prognostic tool for knee pain in adolescence. A very important area. I am very pleased to see the incorporation of psychosocial aspect in the pain. I was wondering about the use of the word Ængstelig/deprimeret (anxious/depressed). Do children aged 8 and a bit older understand that word? Would it be better to use the word bekymret (worried) instead? In the statistics section; you write that you will stratify by traumatic/non-traumatic onset. Why do they choose to do so? I think this paper is well written and described and I therefore have no further comments. I am looking forward the seeing the results. Response: We thank the reviewer for the valuable feedback provided. We have addressed the comments, and individual responses for each specific comment are outlined below. I was wondering about the use of the word Ængstelig/deprimeret (anxious/depressed). Do children aged 8 and a bit older understand that word? Would it be better to use the word bekymret (worried) instead? We thank the reviewer for the insightful comment.The item used to ask about the psychological status was taken from the validated Danish version of the EQ-5d. However, because of the same concern regarding understanding the word Ængstelig/deprimeret, we asked children and adolescents during the cognitive interviews if they had any problems with this word. As it was difficult for some of the younger children to understand this word, we decided to add a note explaining what Ængstelig/deprimeret means. The note was “Ængstelig/deprimeret svarer til at være ked af det (det handler om hvordan du har det, og ikke nødvendigvis på grund af dine smerter)” (In English, anxious/depressed corresponds to be sad – it’s about how you feel and not necessarily because of your knee pain). After adding the note, children did not have further problems with the word. In the statistics section; you write that you will stratify by traumatic/non-traumatic onset. Why do they choose to do so? We thank the reviewer for the insightful comment.The reason why we decided to stratify analysis by traumatic/non-traumatic onset is that previous research has indicated clear differences in the prognosis depending on the type of knee pain onset in both children and adults (please see references below). If the stratification will provide very different prognostic results, it might be possible to use this factor for a quick initial discrimination between low risk vs. medium/high-risk of a bad prognosis. References:El-Metwally A. Lower Limb Pain in a Preadolescent Population: Prognosis and Risk Factors for Chronicity--A Prospective 1- and 4-Year Follow-up Study. Pediatrics. 2005;116(3):673-681. doi:10.1542/peds.2004-1758Belo JN, Berger MY, Koes BW, Bierma-Zeinstra SMA. Prognostic factors in adults with knee pain in general practice. Arthritis Care Res. 2009;61(2):143-151. doi:10.1002/art.2441" } ] }, { "id": "59038", "date": "27 Jan 2020", "name": "Paul Campbell", "expertise": [ "Reviewer Expertise Psychology", "Psychometrics", "Epidemiology", "Musculoskeletal" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMany thanks for the opportunity to review this protocol paper that reports on the rationale and methodology of testing an adolescent knee pain prognostic tool. The protocol is generally well written, and the proposed project to develop a prognostic tool is much needed within the child and adolescent musculoskeletal community. However I do feel some points within the paper require greater clarity and have set out a number of recommendations that I feel would improve the content and presentation of this paper.\nWhilst the authors have aptly discussed the rationale for a prognostic tool within the introduction and briefly touch upon stratified care, I feel they need also mention what this means in terms of aligned treatments based on the risk (i.e. the pathway for stratified patients), a little bit more on this would benefit the introduction.\n\nIn the exclusion criteria the authors mention participants will be excluded if they have an inability to understand the questionnaire, is this a language issue, or for those with learning difficulties, or both, I think the authors might wish to expand on this a little more to give examples.\n\nThe authors state that both parents will be approached before the consultation, does that then require that both parents be present as this won’t often be the case (parent working, single parent etc), also more fundamental is the issue of “before the consultation”, if this is the case how do they know that the knee pain is not referred pain from the spine for example or that whilst the child/adolescent has knee pain that they may also discuss other issues within the consultation and the focus is on those other issues? I might add that the child may be looked after by someone who is not the parent (family member) and perhaps the term “caregiver” should also be used when the term parent is used?\n\nThe authors describe complementary recruitment strategies to work alongside primary care recruitment, I think the use of wider recruitment strategy is good considering the low participation rate of children within primary care to MSK studies. However, whilst the authors have stipulated that children/adolescents will be reminded of the premise for participation, i.e. that participants have been seen by their GP for knee pain, how will this be confirmed? Furthermore, the authors have not explained the timeline for this, for example the recruitment within GP practices is at the time of consultation, however social media recruitment does not state a criteria of when they should have consulted. One of the key prognostic markers for adult MSK pain is time since onset, here you may have potentially two distinct groups, one who have consulted (not clear whether this will be the first consultation or a subsequent visit), and those via social media at presumably post consultation (but not stipulated as to what time period criteria is acceptable, is it a week, month, year etc), this presents issues of case mix, and at the very least the authors would have to collect information about timing of consultations (and timing of when patient first experienced pain) to factor into their analysis.\n\nI think the offer of cinema tickets is a good incentive, however would it not be better to offer a family ticket or at least two tickets as a child would most likely go on their own?\n\nFor the data collection section, I wonder if a diagram might be helpful here as it is not fully clear, does the GP collect the data post consultation, will they (GPs) have time to do this, the authors mention previously that the child/adolescent will be approached to complete the tool before consultation, so does this mean they fill in the tool before consultation and then other information post consultation? The authors should be aware that pre-consultation may reveal different responses to a post-consultation (e.g. reductions in fear avoidance for parent/child due to reassurance from GP etc), this point also relates to point 4 above where alternative recruitment strategies may be used that could lead to different responses due to different timings?\n\nWhen discussing the outcomes, the authors describe that outcomes will be collected by a combination of self-report or parent report, firstly this doesn’t make sense because of the inclusion of “or” as that implies either, whereas it is described as a combination. Does this mean that there will be a choice in who fills in the questionnaire, should it not be self-report AND parent report, or is this related to age and access to online social media platforms where parents/caregivers would have to complete, I think this needs to be explained, and again the researchers should collect information on who fills in the questionnaire as there are differences between proxy and self-report.\n\nParticipant timeline, is table 1 needed, I think it is clear that there will be an assessment at baseline and follow up at 3 months and 6 months for each participant, I don't think a diagram is needed for that? I feel a recruitment flow diagram would be a much better inclusion for this paper.\n\nThe authors discuss the development of the items for the tool, I think a little more should be said about this, for example is this the information within the review (ref 31) or additional literature, also as this is a generation of the candidate items for a prognostic tool it would be good to give some detail (e.g. how many studies within the review, what sort of populations, were they all prospective studies etc). In addition the authors discuss the situation where multiple items or multiple scales were present for items, I feel this whole sentence could do with expansion, what is meant by multiple items and multiple scales, do you mean prognostic factors that have been measured suing multiple items/scales? Give some examples and how a multiple scale construct is then reduced to a single question, how was this done?\n\nWithin the study piloting section, the authors mention the process of improving face, construct, content validity, stressing the importance of this should a GP make an inaccurate diagnosis, I have some concern about this statement as it suggests that the questions asked about pain via the tool are in some way involved in diagnosis, this is surely not the case, can the authors explain what they mean?\n\nIn the stability of the tool section the authors state that children were referred to primary care because of their knee pain, who is it then who refers children to primary care, what is the process for this as this may be a factor to be considered (e.g. how long does it take for a person to be referred to primary care, who does the referral, how are decisions made on a referral etc), I thought it was that the child/parent/caregiver attends primary care?\n\nI am slightly confused about the test-retest parameters, from what is written it appears that the test was done pre and post consultation (i.e. within the same day, perhaps within the same hour), this timeline is too short, the point of test/retest is to ensure that the participants would not remember the responses to the questions asked, so for example the next week, to then ensure that the responses are consistent, I am not convinced this current test stacks up, and any differences would likely be via information received within the consultation (influencing different responses) rather than the actual difference coming from inconsistency in the measure (for example a GP offering reassuring information).\n\nI feel for the general readership it would be helpful to have the tool in English within the paper, and when I tracked down the English version I could see it has 12 questions, whereas the one in the current paper has 13 questions?\n\nThe secondary outcome is the same as the primary outcome, with the only difference being time, could the authors clarify whether this is actually a secondary outcome or just a different time period for the same primary outcome measure?\n\nThe authors state that the statistical analysis plan can be found elsewhere, this protocol paper should include the full statistical analysis plan rather than have a signpost to another protocol, can the authors include the full details in this paper (and if they have then they don’t need to reference another protocol).\n\nThe authors discuss potential floor and ceiling effects, can the authors say a little more about this as presumably some risk items would only identify those at extremes (i.e. at risk) in any case? This is particularly true of items that can identify those at high risk, they may only account for a small proportion of the population with a number of items giving an additive high risk score?\n\nWill the authors examine both each individual item but also within a model (multivariable) to test the independent effects of each item as some many have overlapping qualities (will they also examine redundant items); also will the authors consider model fit (e.g. explained variance) within their analysis?\n\nPoint 5 in the statistical plan is not explained well, could the authors say a little more about the identification of cut off scores and what criteria will be used to decide on this (i.e. are these independently generated from other sample or this one, what determines a poor outcome, if determined from this cohort it will make external validity more problematic), also if more will be given to sensitivity of the tool, how will this factor in the development of risk groups? Also it might be beneficial for the authors to compare risk groups in terms of the baseline characteristics and test across those to give further discriminant information, and in addition it might be worth considering construct validity by comparing to any existing measures, for example this measure could be compared to the generic PPST?\n\nThe discussion might benefit more by describing the potential for the tool in terms of the assistance to clinicians, for example within the study will the research consider how clinicians feel about the tool and whether they feel it would be useful, also what is not clear at present is the potential pathways that are available as defined by risk, do the proposed risk groups have an aligned pathway already for example, if so what might that look like and if not how will these be developed?\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "5238", "date": "21 Feb 2020", "name": "Alessandro Andreucci", "role": "Author Response", "response": "Dear Editorial Team, F1000 Research, We were delighted that the Reviewers of our recent submission \" The Adolescent Knee Pain (AK-Pain) prognostic tool: protocol for a prospective cohort study\" (ID: 21740) provided us with their very helpful feedback on our paper. This has given us the opportunity to apply changes that have led to an improvement of the paper. Reviewer 1 Many thanks for the opportunity to review this protocol paper that reports on the rationale and methodology of testing an adolescent knee pain prognostic tool. The protocol is generally well written, and the proposed project to develop a prognostic tool is much needed within the child and adolescent musculoskeletal community. However I do feel some points within the paper require greater clarity and have set out a number of recommendations that I feel would improve the content and presentation of this paper. Response: We thank the reviewer for the valuable feedback provided. We have addressed the comments and changed the manuscript (changes are underlined in the text), which we feel is now improved. Individual responses for each specific comment are outlined below.1. Whilst the authors have aptly discussed the rationale for a prognostic tool within the introduction and briefly touch upon stratified care, I feel they need also mention what this means in terms of aligned treatments based on the risk (i.e. the pathway for stratified patients), a little bit more on this would benefit the introduction.Response: We thank the reviewer for the comment and have now amended the introduction by providing more information on the aligned treatments based on the risk.The text (Introduction section) now reads “One potential option to support clinical decision making is to develop decision aids such as prognostic tools. These tools often consist of items with prognostic value that can be asked during the clinical consultation or completed prior to the consultation. Thereby patients can be stratified depending on their prognosis (19) and subsequently the best targeted treatment based on the prognostic profile can be offered the patient. In the case of a child or adolescent with knee pain, different treatments or recommendations (e.g. short education session, modification of physical activity levels, exercise, use of painkillers, referral to a specialist) might be provided depending on the risk of a poor prognosis. Examples of prognostic tools that have already been developed include the Keele STarT Back Screening Tool (SBST) for lower back pain in adults (20) or the Pediatric Pain Screening Tool (PPST) for general pediatric pain (19).”2. In the exclusion criteria the authors mention participants will be excluded if they have an inability to understand the questionnaire, is this a language issue, or for those with learning difficulties, or both, I think the authors might wish to expand on this a little more to give examples.Response: Participants are meant to be excluded if they have either language issues or learning difficulties.The text (section Methods, Eligibility and recruitment of participants) now reads “Inability to take part in the study due to an inability to understand the questionnaire (i.e. participants who have issues with Danish language or have learning difficulties)” 3. The authors state that both parents will be approached before the consultation, does that then require that both parents be present as this won’t often be the case (parent working, single parent etc), also more fundamental is the issue of “before the consultation”, if this is the case how do they know that the knee pain is not referred pain from the spine for example or that whilst the child/adolescent has knee pain that they may also discuss other issues within the consultation and the focus is on those other issues? I might add that the child may be looked after by someone who is not the parent (family member) and perhaps the term “caregiver” should also be used when the term parent is used?Response: In Denmark, the general practitioner and his/her staff know in advance if the person is consulting for knee pain based on the booking. However, the general practitioner might also be made aware of the knee pain problem during a consultation regarding other issues. In this case, the questionnaire will be filled out after the consultation. As the reviewer correctly pointed out however, the knee pain might be referred from other body sites (e.g. spine), and we acknowledge that this is a limitation of our study. We have amended the text and made it clearer. The text (section Methods - Eligibility and recruitment of participants) now reads “Participants will be recruited from general practices on the basis of consultation for knee pain. In Denmark, consultations with the GP are booked beforehand, which allows the GP and the GP’s staff to know in advance what the patient is consulting for. The study will be introduced to the caregivers and children before the consultation by a person working at the general practice (e.g. the GP/nurse/secretary). If patients meet the inclusion criteria for being eligible into the study (e.g. patient aged between 8 and 19 years old who consult for knee pain) and agree to participate, they will be provided with the study material (i.e. prognostic tool to be completed). However, if the GP was made aware of the issue of the presence of knee pain during a consultation regarding other health issues, the questionnaire will be completed after the consultation.” 4. The authors describe complementary recruitment strategies to work alongside primary care recruitment, I think the use of wider recruitment strategy is good considering the low participation rate of children within primary care to MSK studies. However, whilst the authors have stipulated that children/adolescents will be reminded of the premise for participation, i.e. that participants have been seen by their GP for knee pain, how will this be confirmed? Furthermore, the authors have not explained the timeline for this, for example the recruitment within GP practices is at the time of consultation, however social media recruitment does not state a criteria of when they should have consulted. One of the key prognostic markers for adult MSK pain is time since onset, here you may have potentially two distinct groups, one who have consulted (not clear whether this will be the first consultation or a subsequent visit), and those via social media at presumably post consultation (but not stipulated as to what time period criteria is acceptable, is it a week, month, year etc), this presents issues of case mix, and at the very least the authors would have to collect information about timing of consultations (and timing of when patient first experienced pain) to factor into their analysis.Response: We thank the reviewer for the very insightful comment.Our plan is to include only those who have consulted within one week. In addition, sensitivity analysis will be applied by comparing results of those recruited in primary care and those recruited through social media to assess potential differences related to case mix. Regarding timing of when patient first experienced pain, this is already assessed with item number 4 of the prognostic tool. We have amended the text and made it clearer. The text (section Methods - Eligibility and recruitment of participants) now reads “Complementary recruitment strategies that might be applied to maximize the recruitment rate are the advertisement of the study through social media (e.g. Facebook, Twitter, Snapchat) and through explicative posters displayed at the participating general practices. In this case, it will be screened that participants have been seen by their GP within the last week and knee pain was part of the consultation.”5. I think the offer of cinema tickets is a good incentive, however would it not be better to offer a family ticket or at least two tickets as a child would most likely go on their own?Response: Although the idea of offering a family ticket or at least two tickets is good, unfortunately we were limited in our choice by financial constrictions. This is why we can offer only one cinema ticket at baseline and one cinema ticket at follow-up, and unfortunately it is not possible to change this strategy now as the study is already on-going.6. For the data collection section, I wonder if a diagram might be helpful here as it is not fully clear, does the GP collect the data post consultation, will they (GPs) have time to do this, the authors mention previously that the child/adolescent will be approached to complete the tool before consultation, so does this mean they fill in the tool before consultation and then other information post consultation? The authors should be aware that pre-consultation may reveal different responses to a post-consultation (e.g. reductions in fear avoidance for parent/child due to reassurance from GP etc), this point also relates to point 4 above where alternative recruitment strategies may be used that could lead to different responses due to different timings?Response: We thank the reviewer for the very insightful comment.Data collection will occur before the consultation and will be facilitated by the general practitioner’s staff (e.g. nurse, secretary), with the tool to be fully completed before consultation. However, as pointed out at point 3 above, the knee pain problem might not be cited as the primary reason for consultation, and the GP only made aware of it during the consultation. In this case, the tool will be completed after the consultation. Being aware of the potential differences in parameters pre-consultation vs. post-consultation we assessed the stability of the tool, which showed good stability of the tool parameters before and after consultation with the general practitioner, as described in section “Methods – Stability of the tool”. 7. When discussing the outcomes, the authors describe that outcomes will be collected by a combination of self-report or parent report, firstly this doesn’t make sense because of the inclusion of “or” as that implies either, whereas it is described as a combination. Does this mean that there will be a choice in who fills in the questionnaire, should it not be self-report AND parent report, or is this related to age and access to online social media platforms where parents/caregivers would have to complete, I think this needs to be explained, and again the researchers should collect information on who fills in the questionnaire as there are differences between proxy and self-report.Response: We thank the reviewer for the comment. The outcome will be collected by questionnaires that will be sent through an e-mail with a link to the web application for online surveys or text message. Within the questionnaire, there are also questions about name, surname and contact information (e-mail and phone number) and about who the person replying to the questionnaire is (i.e. child alone/caregiver/child together with the caregiver). This allows us to know if the person replying is the caregiver or the child/adolescent.We have amended the text and made it clearer. The text (section Methods – Data collection) now reads: “Outcomes will be collected by questionnaires. The adolescent can choose to do this self-reported or caregiver-reported (through an e-mail with a link to the web application for online surveys or text message) at follow-up (two time points: 3-month and 6-month follow-up). The questionnaire also includes a question about who the person replying to the questionnaire is (i.e. child alone/caregiver/child together with the caregiver)” 8. Participant timeline, is table 1 needed, I think it is clear that there will be an assessment at baseline and follow up at 3 months and 6 months for each participant, I don't think a diagram is needed for that? I feel a recruitment flow diagram would be a much better inclusion for this paper.Response: We have replaced table 1 with a recruitment flow diagram and modified the text, which now reads “The process of recruitment and retainment of participants in the study (data collection start date: July 2019 – end date: June 2020) is described in the following flow diagram (figure 1).” 9. The authors discuss the development of the items for the tool, I think a little more should be said about this, for example is this the information within the review (ref 31) or additional literature, also as this is a generation of the candidate items for a prognostic tool it would be good to give some detail (e.g. how many studies within the review, what sort of populations, were they all prospective studies etc). In addition the authors discuss the situation where multiple items or multiple scales were present for items, I feel this whole sentence could do with expansion, what is meant by multiple items and multiple scales, do you mean prognostic factors that have been measured suing multiple items/scales? Give some examples and how a multiple scale construct is then reduced to a single question, how was this done?Response: We thank the reviewer for the insightful comment.Items to be included in the prognostic tool were selected based on a combination of those initially identified with the review (31), of the wider literature on child and adolescent musculoskeletal pain and from meta-analysis of individual participant data (PROSPERO ID CRD42019116861). This allowed us to include also other potential factors that were not previously assessed in the studies identified in the review but were found to be associated with the prognosis of general musculoskeletal pain in other studies and might therefore result as significant contributors to the prognostic model. Regarding the situation where multiple items or multiple scales were present for items relative to the prognostic factors, single items were selected from the most appropriate scales. We have now modified the text (Section Methods - Development of the tool) and made it clearer. The text now reads: “Prognostic factors for knee pain in children and adolescents that can be measured in the context of general practice were identified from a review of the current literature on the topic (29). The review included 26 prospective studies, of which 4 focused on knee pain. These 4 studies included schoolchildren 12-19 years old from Denmark (3 studies) and 10-12 years old from Finland. Initially, the most important domains for the prognosis of knee pain and the specific items to be included in the tool were selected based on the review but also from strength of association from previous studies identified within the literature and from meta-analysis of individual participant data. Prognostic factors for knee pain identified within the review (29) were increasing age, pain frequency, practicing sport more than 2 times/week and low quality of life (second question of item 9 within the tool). Other factors that were identified from the wider literature and meta-analysis of individual participant data (PROSPERO ID CRD42019116861) were knee pain characteristics (pain duration, traumatic/non-traumatic pain onset, limitations in daily activities due to knee pain, presence of knee pain in one knee or both knees), presence of pain in other body sites, gender, sleep, smoking, psychological factors, parental history of pain. During this stage, great emphasis was given to include only the most necessary factors considering the ultimate use of the prognostic tool (i.e. it should be used in a reasonable time of a consultation within general practice). Items relative to the specific prognostic factors were initially selected from validated scales when possible (e.g. regarding pain characteristics, limitation in daily activities, sport participation, psychological factors, sleep), or from previous studies. When multiple items within a scale or multiple scales were available for a prognostic factor, the relevant literature on the topic was identified and discussed at meetings with GPs and staff working at the Center of General Practice at Aalborg University in order to select the most appropriate items within the possible options. For example, the sleep item was selected from the self-reported Pediatric Quality of Life Inventory, one question of the psychological factor item from the Pain Catastrophizing Scale for Children, one question of the psychological factor item and the item relative to limitations in daily activities from the EuroQol Five-Dimensional Questionnaire Youth version.” 10. Within the study piloting section, the authors mention the process of improving face, construct, content validity, stressing the importance of this should a GP make an inaccurate diagnosis, I have some concern about this statement as it suggests that the questions asked about pain via the tool are in some way involved in diagnosis, this is surely not the case, can the authors explain what they mean?Response: The aim of the study piloting was to improve the comprehensibility and wording of the questions included in the tool, so that adolescents would provide information about the parameters related to the knee pain prognosis that were as close as possible to the real values and therefore limit potential misreporting. As the reviewer correctly pointed out, the tool is not conceived as a replacement of the diagnosis made by the general practitioner, but only as a support for understanding the prognosis together with the clinical examination. We have now modified the text and made it clearer. The text (section Methods – Study piloting) now reads: “The aim was to improve the face, construct and content validity of the tool at this stage. This is especially important considering that a worse outcome can result if there is a lack of communication between the GP and the child about the knee pain characteristics and the factors related to knee pain assessed within the tool (33).”11. In the stability of the tool section the authors state that children were referred to primary care because of their knee pain, who is it then who refers children to primary care, what is the process for this as this may be a factor to be considered (e.g. how long does it take for a person to be referred to primary care, who does the referral, how are decisions made on a referral etc), I thought it was that the child/parent/caregiver attends primary care?Response: We have amended the imprecision in the text, as we actually meant the child/adolescent who consult primary care for knee pain. We have modified the text, which now reads “Children and adolescents who consulted primary care and their caregivers were given the prognostic tool (together with the informed consent) to be completed in the waiting room of the general practice before the consultation.”12. I am slightly confused about the test-retest parameters, from what is written it appears that the test was done pre and post consultation (i.e. within the same day, perhaps within the same hour), this timeline is too short, the point of test/retest is to ensure that the participants would not remember the responses to the questions asked, so for example the next week, to then ensure that the responses are consistent, I am not convinced this current test stacks up, and any differences would likely be via information received within the consultation (influencing different responses) rather than the actual difference coming from inconsistency in the measure (for example a GP offering reassuring information).Response: We thank the reviewer for the comment and acknowledge the inaccuracy in the language used within the text. As pointed out by the reviewer, the GP might influence the perception of certain parameters related to pain (i.e. psychological factors, pain duration) through the discussion occurring during the consultation. In order to test if responses to the questions asked were influenced by the consultation with the GP (and to estimate the difference in responses), the stability of the tool before and after the consultation was tested and results showed good stability. However, as the reviewer correctly pointed out, this cannot be considered a test-retest as the time frame between the two measurements was too short. We have amended the text, (Section Methods – Stability of the tool) which now reads “A pilot study to assess the stability of the prognostic tool (31) in children and adolescents pre- and post- consultation was carried out.”13. I feel for the general readership it would be helpful to have the tool in English within the paper, and when I tracked down the English version I could see it has 12 questions, whereas the one in the current paper has 13 questions?Response: The English version, with 13 questions is accessible at: https://doi.org/10.7910/DVN/QKWOOT 14. The secondary outcome is the same as the primary outcome, with the only difference being time, could the authors clarify whether this is actually a secondary outcome or just a different time period for the same primary outcome measure?Response: We thank the reviewer for the comment and have amended the imprecision in the text. In our study, the outcome is the same is collected at two endpoints. We decided to consider the knee pain prognosis at 3-month as the primary endpoint and the knee pain prognosis at 6 months as a secondary endpoint.We have modified the text (Section Methods – Outcome), which now reads “The outcome measure will be the recurrence/persistence of activity-limiting knee pain (i.e. defined as participants reporting yes to having pain that is limiting activities in the same knee) at follow-up (28). Participants will be asked about continuity of their knee pain (i.e. “do you still have knee pain?” and, if they reply “no”, “when did your stop having knee pain?”), to enable the distinction between recurrence (on/off knee pain episodes between baseline and follow-up) and persistence (continuous knee pain from baseline to follow-up) of knee pain. The primary end-point that will be collected is the recurrence/persistence of activity-limiting knee pain at 3-month follow-up, while the secondary end-point is the recurrence/persistence of activity-limiting knee pain at 6-month follow-up” 15. The authors state that the statistical analysis plan can be found elsewhere, this protocol paper should include the full statistical analysis plan rather than have a signpost to another protocol, can the authors include the full details in this paper (and if they have then they don’t need to reference another protocol).Response: We have now corrected the text (section Methods – Statistics), inserted the full analysis plan within this paper and deleted the reference to the other protocol. 16. The authors discuss potential floor and ceiling effects, can the authors say a little more about this as presumably some risk items would only identify those at extremes (i.e. at risk) in any case? This is particularly true of items that can identify those at high risk, they may only account for a small proportion of the population with a number of items giving an additive high risk score?Response: We thank the reviewer for the very insightful comment. As correctly pointed out by the reviewer, for items that represent an ordinal or categorical variable with more than two potential response categories (for example 5 categories), the responses given might be only the top or bottom extreme of the scales. For example, this might happen for items related to psychological factors and sleep. In this case, participants with higher levels of anxiety/depression, helplessness or sleep problems would most likely fall in the high-risk category. A potential option to account for this is to initially test the independent effect of each item by means of multiple logistic regression, include those items that show a significant contribution to the model and subsequently apply different weights based on the strength of association. For example, a combination of the different items can be used to identify the scores for defining the risk groups (i.e. low/medium/high) and if items that identify only a small proportion of the population (e.g. high levels of psychological factors or sleep problems) are associated with a high risk of a bad prognosis, they can be selected and used to give an additive high-risk score. 17. Will the authors examine both each individual item but also within a model (multivariable) to test the independent effects of each item as some many have overlapping qualities (will they also examine redundant items); also will the authors consider model fit (e.g. explained variance) within their analysis?Response: We thank the reviewer for the comment. The plan is to test the independent effect of each item by means of multiple logistic regression and include only those items that show a significant contribution to the model. Therefore, the items that will not show a significant contribution will be excluded. In addition, items might be weighted differently based on the strength of association. We have modified the text (section Methods – Statistics), which now reads: “We will estimate the knee pain prognosis(i.e. recurrence/persistence of knee pain, dichotomous outcome) at 3-month and 6-month follow-up by means of multiple logistic regression to estimate ORs and 95% confidence intervals for each item included in the tool. This allows to assess the independent effects of each item and will inform on which factors are most related to the prognosis of knee pain (only the items that will show a statistically significant contribution to the model will be selected). This will also provide an insight on the scores of the prognostic tool (both overall and for subscales) to be applied for the creation of the initial risk groups. Alternatively, the RR will be estimated if another linear model analysis will be carried out. In addition, a potential option is to apply different weights to the items based on the strength of association.” 18. Point 5 in the statistical plan is not explained well, could the authors say a little more about the identification of cut off scores and what criteria will be used to decide on this (i.e. are these independently generated from other sample or this one, what determines a poor outcome, if determined from this cohort it will make external validity more problematic), also if more will be given to sensitivity of the tool, how will this factor in the development of risk groups? Also it might be beneficial for the authors to compare risk groups in terms of the baseline characteristics and test across those to give further discriminant information, and in addition it might be worth considering construct validity by comparing to any existing measures, for example this measure could be compared to the generic PPST?Response: We thank the reviewer for the insightful comment and provide some clarifications on this point.At each follow-up time-point, a poor outcome is defined as the presence of activity-limiting knee pain. As the cut-off scores will be generated by using data of this sample, the prognostic model developed within this cohort will have to be externally validated with another cohort in the future. Regarding sensitivity, we stated that more importance will be given to sensitivity of the tool, meaning that we will choose cut-off scores for the definition of risk groups that will allow to identify the majority of those with a potential bad prognosis in the high risk group, and rule out from the group those with a good prognosis. In addition, despite comparing the prognostic tool to the pediatric pain screening tool might be an option, it should be taken into account that the pediatric pain screening tool was validated in a tertiary care clinic and may operate differently in a primary care setting, which is where our prognostic tool is meant to operate. Therefore, the comparison might result in a poor match between risk group categories of the two tools due to differences in the severity of the sample used to develop the tools. We have modified the text, which now reads: “Data from this sample will be used for the creation of risk groups for the recurrence/persistence of knee pain, on the basis of cut-off scores identified using the ROC curves. Weights based on the strength of association identified with the multiple logistic regression might be applied. The initial idea is to have two or three risk groups (e.g. low/medium/high), which have to be clinically meaningful. More importance will be given to the sensitivity of the tool over the specificity. This means that in the presence of different cut-off scores for the inclusion of patients in the high-risk group, the cut-off that will allow to identify the majority of those with a bad prognostic outcome will be chosen. This will be done to avoid the misclassification of patients at high-risk in the medium or low-risk group.” 19. The discussion might benefit more by describing the potential for the tool in terms of the assistance to clinicians, for example within the study will the research consider how clinicians feel about the tool and whether they feel it would be useful, also what is not clear at present is the potential pathways that are available as defined by risk, do the proposed risk groups have an aligned pathway already for example, if so what might that look like and if not how will these be developed?Response: We thank the reviewer for the insightful comment and provide some clarifications on this point.There are other steps that will follow the development of this tool. As the reviewer highlights, there are additional steps to be completed before implementing the tool. This includes 1) an external validation of the tool 2) developing the matched care pathways 3) mixed-methods studies to understand how the tool should look and operate in the context of general practice, and 4) subsequent clinical trials to test the impact of the tool. A cluster randomized controlled trial will be performed where patients will be randomized to stratified care provided by using the tool or to the best current care, and differences in clinical outcomes will be assessed. The original idea is to provide targeted treatments to the three risk groups. For example, patients in the low risk group might be provided with an initial short patient education session and given a leaflet that reinforces the information given by the GP. For the medium risk group, strengthening exercises and load management might be delivered, while for the high-risk group a tailored treatment might be delivered that will target also psychological factors. However, these are only initial suggested treatments, and additional treatments might be developed depending on the items that will prove to be significantly associated with a bad prognosis (e.g. potential further treatments might be advices regarding having a good sleep hygiene). Regarding assistance to the clinician, this tool together with another tool for the knee pain diagnosis, will be part of a package (decision tool) that will assist clinicians in the diagnosis and treatment of knee pain. Future qualitative studies will be needed to assess the GPs´ behaviour when using the tool. We have now modified the text and added this part to the section \"Use of the tool for providing stratified care\":“The development of this tool fits within a wider research program, which overall aim is to provide a stratified approach to primary care management of child and adolescent knee pain that can result in clinical and economic benefits compared with current best practice. Therefore, future perspectives include the use of this tool in a randomized controlled trial, which will investigate whether subgrouping patients using the tool, combined with targeted treatment, is more clinically effective (i.e. it will reduce long- term disability from knee pain) and cost-effective compared to best current care. In addition, there is scope for performing future qualitative studies to assess the GPs´ behavioral change when using the tool (e.g. changes in referral to physical therapy, diagnostic tests and medication prescriptions)” Reviewer 2 Thank you for giving me the opportunity to review this well described paper about development and test of a prognostic tool for knee pain in adolescence. A very important area. I am very pleased to see the incorporation of psychosocial aspect in the pain. I was wondering about the use of the word Ængstelig/deprimeret (anxious/depressed). Do children aged 8 and a bit older understand that word? Would it be better to use the word bekymret (worried) instead? In the statistics section; you write that you will stratify by traumatic/non-traumatic onset. Why do they choose to do so? I think this paper is well written and described and I therefore have no further comments. I am looking forward the seeing the results. Response: We thank the reviewer for the valuable feedback provided. We have addressed the comments, and individual responses for each specific comment are outlined below. I was wondering about the use of the word Ængstelig/deprimeret (anxious/depressed). Do children aged 8 and a bit older understand that word? Would it be better to use the word bekymret (worried) instead? We thank the reviewer for the insightful comment.The item used to ask about the psychological status was taken from the validated Danish version of the EQ-5d. However, because of the same concern regarding understanding the word Ængstelig/deprimeret, we asked children and adolescents during the cognitive interviews if they had any problems with this word. As it was difficult for some of the younger children to understand this word, we decided to add a note explaining what Ængstelig/deprimeret means. The note was “Ængstelig/deprimeret svarer til at være ked af det (det handler om hvordan du har det, og ikke nødvendigvis på grund af dine smerter)” (In English, anxious/depressed corresponds to be sad – it’s about how you feel and not necessarily because of your knee pain). After adding the note, children did not have further problems with the word. In the statistics section; you write that you will stratify by traumatic/non-traumatic onset. Why do they choose to do so? We thank the reviewer for the insightful comment.The reason why we decided to stratify analysis by traumatic/non-traumatic onset is that previous research has indicated clear differences in the prognosis depending on the type of knee pain onset in both children and adults (please see references below). If the stratification will provide very different prognostic results, it might be possible to use this factor for a quick initial discrimination between low risk vs. medium/high-risk of a bad prognosis. References:El-Metwally A. Lower Limb Pain in a Preadolescent Population: Prognosis and Risk Factors for Chronicity--A Prospective 1- and 4-Year Follow-up Study. Pediatrics. 2005;116(3):673-681. doi:10.1542/peds.2004-1758Belo JN, Berger MY, Koes BW, Bierma-Zeinstra SMA. Prognostic factors in adults with knee pain in general practice. Arthritis Care Res. 2009;61(2):143-151. doi:10.1002/art.2441" } ] } ]	1	https://f1000research.com/articles/8-2148
https://f1000research.com/articles/8-1860/v1	06 Nov 19	{ "type": "Research Article", "title": "Delayed administration of recombinant plasma gelsolin improves survival in a murine model of severe influenza", "authors": [ "Zhiping Yang", "Alice Bedugnis", "Susan Levinson", "Mark DiNubile", "Thomas Stossel", "Quan Lu", "Lester Kobzik", "Zhiping Yang", "Alice Bedugnis", "Susan Levinson", "Mark DiNubile", "Thomas Stossel", "Quan Lu" ], "abstract": "Background: Host-derived inflammatory responses contribute to the morbidity and mortality of severe influenza, suggesting that immunomodulatory therapy may improve outcomes. The normally circulating protein, human plasma gelsolin, is available in recombinant form (rhu-pGSN) and has beneficial effects in a variety of pre-clinical models of inflammation and injury.\n\nMethods: We evaluated delayed therapy with subcutaneous rhu-pGSN initiated 3 to 6 days after intra-nasal viral challenge in a mouse model of influenza A/PR/8/34. Results: Rhu-pGSN administered starting on day 3 or day 6 increased survival (12-day survival: 62 % vs 39 %, pGSN vs vehicle; p < 0.00001, summary of 18 trials), reduced morbidity, and decreased pro-inflammatory gene expression. Conclusions: Rhu-pGSN improves outcomes in a highly lethal influenza model when given after a clinically relevant delay.", "keywords": [ "influenza", "pneumonia", "plasma gelsolin", "immunomodulation", "host-directed" ], "content": "Introduction\n\nSeasonal influenza continues to be a cause of substantial morbidity and mortality. There is also a fear that a new virulent influenza strain could cause high death rates, similar to those seen during the 1918 pandemic1. The 2009 pandemic revealed the limitations of available public health interventions and current vaccines2. While some antiviral drugs (e.g., oseltamivir) are currently in use, they suffer from a short time window of efficacy and increasing viral resistance3. Hence, a substantial but unmet need exists for new therapeutic agents, especially for life-threatening infections.\n\nThe pathogenesis of influenza involves dysregulated and injurious host inflammatory responses4–6. This observation suggests that better inflammation control with immunomodulatory therapy may be able to reduce the morbidity and mortality seen in severe infections. Recombinant human plasma gelsolin (rhu-pGSN) is an attractive candidate because it dampens excessive and injurious inflammation and augments antimicrobial defenses. Moreover, it has successfully passed several of the safety, toxicity, and regulatory tests needed to go from ‘bench to bedside’.\n\nGelsolin was first identified in the cytoplasm of macrophages. It was further identified in many vertebrate cells, and is a highly conserved protein with many functions7,8. A unique characterstic of gelsolin at the gene level is the existence of a splice variant which encodes a distinct plasma isoform (pGSN). This isoform is released into extracellular fluids and differs from its cytoplasmic counterpart by the inclusion of an additional 25 amino acids at the N-terminal sequence. Normal mammalian blood contains pGSN at concentrations of 200–300 µg/ml, making it one of the most abundant proteins in plasma.\n\nAmong pGSN’s many functions is to dissolve the actin gels is the one that gave it its name: dissolving the actin gels that arise from cellular debris. The gels have the deleterious property of forming a protective biofilm that reduces the ability of cellular and humoral defenses to access embedded pathogenic organisms. This function leads to accumulation of pGSN at sites of tissue damage. The intereaction with actin reduces pGSN’s binding to and inactivation of a host of microbial toxins and inflammatory mediators (for example, lysophosphatidic acid, sphingosine-1-phosphate, platelet-activating factor, fibronectin, endotoxin and lipoteichoic acid). The local dynamic balance of these mediators can modulate host defense9,10. A final noteworthy function of pGSN is its ability to augment the phagocytosis and killing of both Gram-positive and -negative bacteria by macrophages11. By stripping actin off macrophage scavenger receptors, pGSN promotes phagocytosis. It also enhances killing by stimulating the constitutive NOS3 enzyme system11,12. As the acute injury subsides, pGSN is free to bind and inhibit inflammatory substances, promoting resolution of injury at the infectious site. The local capture of pGSN by exposed actin reduces the levels of pGSN in the circulation commensurate to the magnitude of tissue injury. The relative abundance ofpGSN typically allows it to render inactive any pro-inflammatory mediators that enter the systemic circulation and helps to prevent organ damage distant from the injury site. In severe infection, systemic depletion of pGSN can result in loss of its protective effects. Indeed, there is a robust correlation between how much pGSN levels decrease and probability of mortality. As might be predicted from these observations, systemic treatment withpGSN has reduced pathologic changes and mortality in numerous preclinical animal disease models7,13,14.\n\nRelevant to the severe pneumonia seen in fatal influenza, administration of rhu-pGSN improved survival in murine primary or secondary (post-influenza) pneumococcal pneumonia, a benefit seen without any antibiotic treatment11,15. These results have established proof-of-principle for the potential benefit of rhu-pGSN for bacterial pneumonias, including the secondary pneumonias often found as a complication of influenza. Here we report that rhu-pGSN improves outcomes in a mouse primary influenza model without superimposed bacterial infections.\n\n\nMethods\n\nAll protocols were approved by the Harvard Medical Area Biosafety and Animal Care and Use Committees.\n\nNormal 6- to 8-week-old male CD1 mice were obtained from Charles River Laboratories (Wilmington, MA). A murine-adapted strain of H1N1 influenza virus, A/Puerto Rico/8/1934 (PR8), quantified as plaque-forming units (PFU) was procured from ViraSource (Durham, NC). Mice were anesthetized with 72 mg/kg ketamine plus 9.6 mg/kg xylazine administered via intraperitoneal injection. Mice then received an intranasal instillation of 25 μl suspension of PBS containing virus (ranging from 400–1000 PFU depending on the trial) or vehicle alone. Initial titration identified 400 PFU as a dose that led to ~60% mortality in vehicle-treated mice, and this dose was used in a majority of the trials (see Table 1). Most trials used at least 10 mice per group for the vehicle and pGSN treatment groups; details of the influenza dose, total number of mice, and their weights are provided in the tables in Underlying Data16.\n\n* Treatment benefit scored as Yes if % survival ≥10% better with pGSN vs. Vehicle; No if % survival <10% better with pGSN.\n\nRecombinant human pGSN (rhu-pGSN) was synthesized in E. coli and purified by Fujifilm Biosynth (Billingham, UK). Rhu-pGSN was administered daily to mice by subcutaneous injection starting on day 3 or 6 after infection, at doses ranging from 0.5–5 mg as detailed in the Results. We monitored the mice for 12 days, measuring survival, changes in weight and overall morbidity using a composite index (i.e., 1 point each for hunched appearance, ruffled fur or partly closed eyes; 1.5 points for prolapsed penis or splayed hind quarter; 2 points for listlessness, with a maximum score of 8; the assessment was performed without blinding to treatment group) adapted from guidelines described previously17. Weights and morbidity scores for the last day alive were carried forward for animals that did not survive.\n\nLung tissue was obtained on days 7 and 9 after infection from mice treated with either vehicle or rhu-pGSN (dosed 2 mg per day starting on day 3 after infection, then increased to 5 mg per day on day 7). RNA was isolated using the RNAEasy mini-kit (Qiagen, Germantown, MD) according to manufacturer’s instructions. RNA samples were analyzed using the Mouse DriverMap targeted gene expression profiling panel from Cellecta (Mountain View, CA). The Cellecta platform uses highly multiplexed RT-PCR amplification and next-generation sequencing (NGS) quantitation to measure expression of 4753 protein-coding and functionally significant mouse genes. The procedure detailed in the Cellecta User Manual, item 5.3 was followed to create amplified index libraries which were sequenced on a Illumina NextSeq 500 instrument. The sequencing data was converted to FASTQ format and then further analyzed using DriverMap Sample Extraction software. This produces a raw data matrix file of counts for each sample in columns aligned to the 4753 gene panel.\n\nData were analyzed using Prism (GraphPad Software) or SAS (SAS Institute) software. Differences in Kaplan-Meier survival curves were analyzed using a log-rank test with Sidak adjustment for multiple comparisons. A Breslow-Day test for homogeneity of the pGSN versus vehicle comparison across studies yielded p>0.2, indicating homogeneity could not be rejected and supporting the overall comparison across studies, which was carried out via the log-rank (Mantel-Cox) test stratified by trial. For other measurements, differences between groups were examined by ANOVA. The transcriptome profiling results scaled to normalize column counts, were converted to log2 counts (after addition of 0.1 to all cells to eliminate zero values) and then analyzed using Qlucore software (Lund, Sweden). Further analysis of gene set enrichment was performed using tools (Panther version 14.118 and MetaCore (version 19.3, Clarivate Analytics, Philadelphia, PA)) that allow evaluation using a custom background gene list (i.e., the ~4700 genes measured using the Cellecta DriverMap platform).\n\n\nResults\n\nWe tested a variety of dose and timing regimens to evaluate the potential of rhu-pGSN to improve outcomes, conducting a total of 18 trials that are tabulated in Table 1 and summarized in Table 2. To mimic likely clinical usage, mice were not treated until several days post-challenge\n\npGSN, plasma gelsolin\n\nThe main finding was that delayed treatment with rhu-pGSN resulted in significant improvement in the survival of mice (Figure 1). All studies combined yielded 39% (93/236) surviving mice treated with vehicle and 62% (241/389) surviving mice treated with pGSN on day 12 (p = 0.000001, Figure 1A). Improved survival was observed whether the delayed treatment was started on day 6 (Figure 1C) or day 3 after infection (Figures 1E, G). Similarly, compared to vehicle treatment, rhu-pGSN resulted in decreased morbidity scores (Figures 1B, D, F, H). In contrast, no statistically significant difference in weight loss or recovery (in surviving animals) was consistently observed in the experiments summarized in Figure 1. The sole exception was found in the trials testing a dose regimen of initially low (> 2 mg rhu-pGSN on days 3-6/7, then 5 mg through day 11). The latter set of trials led to weights (compared to day 0) at the end of study of 81.4 ± 4.7% in vehicle-treated mice versus 85 ± 2.6% in pGSN-treated mice (p < 0.0001, summary of 4 trials, see also Table 1 and Table 2, and more detailed tabulation of all experiments in Extended data16. A beneficial effect of rhu-pGSN was observed in a majority but not all of the 18 individual trials (Table 1, see Discussion).\n\nComparison of survival rates (A, C, E, G) and morbidity (B, D, F, H) in mice treated with rhu-pGSN or vehicle. (A, B) Results for all 18 trials (typically 10 or more mice per group, see details in Table 1 and Table 2) using delayed treatment. Some trials initiated treatment in different arms on day 6 or day 3. (C, D) Results for 13 trials using delayed treatment starting on day 6 or later. (E, F) Results for eight trials using treatment starting on day 3. (G, H) Results for four trials starting with an initially lower dose on day 3 with an increased dose starting on day 6/7. * = 0.000001, 0.00001, 0.0005, 0.0005 for A, C, E, G, respectively; p < 0.0001 for B, D, F, H.\n\nTo evaluate whether rhu-pGSN treatment modified the transcriptome profile (see Underlying data) of infected lungs, we harvested lung tissue just before (day 7) and after (day 9) the usual onset of mortality (day 8) in this model (n = 5 per group per day). Comparison of lung samples obtained at day 7 showed no significant differences. In contrast, analysis of day 9 samples identified 344 differentially expressed genes in the rhu-pGSN-treated group, comprised of 195 down-regulated and 149 up-regulated genes. The top 50 up- and down-regulated genes are shown in Figure 2, which is notable for the many cytokine and immune-related genes prominent among those down-regulated in the rhu-pGSN-treated group (including IL10, IL12rb, CTLA4, and CCRs9, 7 and 5, among others). We performed gene enrichment analysis of the down-regulated gene list using the Panther online analysis tool to query GO Ontology or Reactome databases. The main findings were a reduction of expression of biological processes linked to immune and inflammatory responses, or release of cytokine and other cellular activators. The top 10 most significant processes/pathways are shown in Table 3. Analysis using a different gene enrichment analysis software tool (MetaCore) produced similar results. Analysis of the up-regulated gene list identified enrichment of processes related to tissue morphogenesis and epithelial/epidermal cell differentiation (consistent with repair of influenza-mediated damage, see Discussion). We present details of the DriverMap gene list, the differentially expressed genes identified, and the full results of gene enrichment analyses using the down- and up-regulated gene lists to query the Panther and MetaCore databases in worksheets 2–15 in a spreadsheet available in Extended data19.\n\nHeat map showing top 50 down-regulated (left) and up-regulated (right) genes in the lungs of rhu-pGSN treated animals on day 9 (range -2 (blue) to + 2 (red)).\n\n\nDiscussion\n\nWe sought to evaluate the potential of rhu-pGSN to improve outcomes in severe influenza using a clinically relevant scenario of delaying initiation of treatment. The key finding was that delayed pGSN treatment significantly improved survival, either when used starting on day 3 or even starting as late as day 6 after infection.\n\nSome limitations merit discussion. The first is the experimental variability we observed and report. Treatment with rhu-pGSN increased survival in a majority of the experiments conducted, but not in all of them. For a subset of the negative trials, we could postulate plausible potential explanations (e.g., technical issues with the viral stock, variation in instillation method, insufficient initial rhu-pGSN dose in the ‘low dose then high dose’ trials). To the extent possible, we adjusted our methods to reduce these potential sources of variability. However, for the remainder of the negative trials we simply do not have a good explanation for the outcome. Hence, we have chosen to present all the data whether positive or negative to provide a full report of the findings.\n\nWe also manipulated the experimental variables, in part to address larger questions (e.g., can treatment as late as day 6 vs day 3 after onset of infection be effective?) and in some cases to explore potential reasons for the intermittent variability in our results (e.g., trial 14 tested the potential influence of differences in initial weight of the mice we used). Ultimately, we observed beneficial effects whether the survival analysis included all the trials (Figures 1A, B) or those using treatment starting at day 6 or day 3 (Figures 1C–H).\n\nNotably, rhu-pGSN did not rescue all of the mice dying from influenza in our model, offering only a partial (albeit significant) survival benefit. Given the goal of identifying a novel therapy for severe influenza, an optimistic interpretation is that this occurred in mice without the supportive fluid and respiratory care given to hospitalized patients, and that similar or more robust benefits might be observed in the clinical setting. The results establish a potential benefit for rhu-pGSN but this potential needs further evaluation in a larger animal model, e.g. ferret20 and then (if results warrant), testing in a clinical trial to determine its role in therapy for severe influenza in human patients. Our findings rely on studies with only one strain of influenza in only one strain of one model species, the mouse. Nevertheless, we favor future experimentation in a larger animal model as the logical next step, rather than further studies in mice. Additional investigations using other influenza or mouse strains would not resolve the suggestion (hope) of possible clinical benefit offered by our results. Hence, large animal experiments deserve priority.\n\nOur study did not address the mechanism(s) for the beneficial action of rhu-pGSN. The available literature identifies numerous inflammatory mediators whose function can be modulated by pGSN (e.g. sphingosine-1-phophate9, endotoxin10, platelet activating factor21). The transcriptome profiling results are consistent with a beneficial down-regulation of the overly exuberant immune and inflammatory response that characterizes severe influenza22–24. Further investigation of the many possible single or combination targets by which pGSN may be acting is warranted. However, a complete delineation of its mechanisms will take substantial effort and time to achieve. If effective, therapeutic use of rhu-pGSN should be pursued even in the absence of a full map of its complex effects. This position reflects in part the fact that pGSN is a normal, abundant protein in human plasma, and has passed initial safety evaluation in human subjects with community acquired pneumonia (personal communication, Levinson, S. & DiNubile, M.). Finally, it is worth speculating that rhu-pGSN treatment may also benefit patients with severe influenza by reducing the risk of the common complication of secondary bacterial pneumonia25,26. This possibility is suggested by other studies from our laboratory, showing rhu-pGSN improved survival of mice with post-influenza bacterial pneumonia11.\n\nIn summary, rhu-pGSN can improve outcomes in a highly lethal murine influenza model when given after a clinically relevant delay. These findings are consistent with the benefits seen in models of pneumococcal pneumonia. The modes of action for pGSN involve host responses and do not seem to depend on the specific type of pathogen. Our findings support further investigation of pGSN as an adjunctive therapy for severe influenza and other viral infections.\n\n\nData availability\n\nHarvard Dataverse: Expanded Tables 1 & 2. https://doi.org/10.7910/DVN/53GJY116.\n\nThis project contains data on each experimental group, as shown in Table 1 and Table 2, with additional variables, such as weight, and statistical analyses.\n\nNCBI Gene Expression Omnibus: Transcriptome profiling of lung tissue from influenza-infected mice treated with plasma gelsolin. Accession number GSE138986; https://identifiers.org/geo:GSE138986.\n\nHarvard Dataverse: Transcriptome analysis of gelsolin vs vehicle treatment in mouse influenza infected lungs. https://doi.org/10.7910/DVN/8HBFD719.\n\nHarvard Dataverse: ARRIVE checklist for ‘Delayed administration of recombinant plasma gelsolin improves survival in a murine model of severe influenza’. https://doi.org/10.7910/DVN/VQBKLF27.\n\nData hosted on Harvard Dataverse are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors thank Dr. James Bolognese for consultation on statistical analysis.\n\n\nReferences\n\nMorens DM, Taubenberger JK: Pandemic influenza: certain uncertainties. Rev Med Virol. 2011; 21(5): 262–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFineberg HV: Pandemic preparedness and response--lessons from the H1N1 Influenza of 2009. N Engl J Med. 2014; 370(14): 1335–42. PubMed Abstract \| Publisher Full Text\n\nLackenby A, Besselaar TG, Daniels RS, et al.: Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors and status of novel antivirals, 2016-2017. Antiviral Res. 2018; 157: 38–46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIwasaki A, Pillai PS: Innate immunity to influenza virus infection. Nat Rev Immunol. 2014; 14(5): 315–28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHerold S, Becker C, Ridge KM, et al.: Influenza virus-induced lung injury: pathogenesis and implications for treatment. Eur Respir J. 2015; 45(5): 1463–78. PubMed Abstract \| Publisher Full Text\n\nGregory DJ, Kobzik L: Influenza lung injury: mechanisms and therapeutic opportunities. Am J Physiol Lung Cell Mol Physiol. 2015; 309(10): 1041–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPiktel E, Levental I, Durnas B, et al.: Plasma Gelsolin: Indicator of Inflammation and Its Potential as a Diagnostic Tool and Therapeutic Target. Int J Mol Sci. 2018; 19(9): pii: E2516. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilacci P, Mazzolai L, Gauci C, et al.: Gelsolin superfamily proteins: key regulators of cellular functions. Cell Mol Life Sci. 2004; 61(19-20): 2614–23. PubMed Abstract \| Publisher Full Text\n\nBucki R, Kulakowska A, Byfield FJ, et al.: Plasma gelsolin modulates cellular response to sphingosine 1-phosphate. Am J Physiol Cell Physiol. 2010; 299(6): C1516–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBucki R, Georges PC, Espinassous Q, et al.: Inactivation of endotoxin by human plasma gelsolin. Biochemistry. 2005; 44(28): 9590–7. PubMed Abstract \| Publisher Full Text\n\nYang Z, Chiou TT, Stossel TP, et al.: Plasma gelsolin improves lung host defense against pneumonia by enhancing macrophage NOS3 function. Am J Physiol Lung Cell Mol Physiol. 2015; 309(1): L11–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrdija CM, Chiou TT-Y, Yang Z, et al.: Free actin impairs macrophage bacterial defenses via scavenger receptor MARCO interaction with reversal by plasma gelsolin. Am J Physiol Lung Cell Mol Physiol. 2017; 312(6): L1018–L1028. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCohen TS, Bucki R, Byfield FJ, et al.: Therapeutic potential of plasma gelsolin administration in a rat model of sepsis. Cytokine. 2011; 54(3): 235–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChristofidou-Solomidou M, Scherpereel A, Solomides CC, et al.: Recombinant plasma gelsolin diminishes the acute inflammatory response to hyperoxia in mice. J Investig Med. 2002; 50(1): 54–60. PubMed Abstract \| Publisher Full Text\n\nYang Z, Bedugnis A, Levinson S, et al.: Delayed Administration of Recombinant Plasma Gelsolin Improves Survival in a Murine Model of Penicillin-Susceptible and Penicillin-Resistant Pneumococcal Pneumonia . J Infect Dis. 2019; 220(9): 1498–1502. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKobzik L: \"Expanded Tables 1 & 2\". Harvard Dataverse, V1 2019. http://www.doi.org/10.7910/DVN/53GJY1\n\nBurkholder T, Foltz C, Karlsson E, et al.: Health Evaluation of Experimental Laboratory Mice. Curr Protoc Mouse Biol. 2012; 2: 145–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMi H, Muruganujan A, Casagrande JT, et al.: Large-scale gene function analysis with the PANTHER classification system. Nature Protocols. 2013; 8(8): 1551–66. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKobzik L: \"Transcriptome analysis of gelsolin vs vehicle treatment in mouse influenza infected lungs\". Harvard Dataverse, V1 2019. http://www.doi.org/10.7910/DVN/8HBFD7\n\nAlbrecht RA, Liu WC, Sant AJ, et al.: Moving Forward: Recent Developments for the Ferret Biomedical Research Model. MBio. 2018; 9(4): pii: e01113-18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOsborn TM, Dahlgren C, Hartwig JH, et al.: Modifications of cellular responses to lysophosphatidic acid and platelet-activating factor by plasma gelsolin. Am J Physiol Cell Physiol. 2007; 292(4): C1323–30. PubMed Abstract \| Publisher Full Text\n\nKalil AC, Thomas PG: Influenza virus-related critical illness: pathophysiology and epidemiology. Crit Care. 2019; 23(1): 258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGounder AP, Boon ACM: Influenza Pathogenesis: The Effect of Host Factors on Severity of Disease. J Immunol. 2019; 202(2): 341–350. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBiondo C, Lentini G, Beninati C, et al.: The dual role of innate immunity during influenza. Biomed J. 2019; 42(1): 8–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShirey KA, Perkins DJ, Lai W, et al.: Influenza \"Trains\" the Host for Enhanced Susceptibility to Secondary Bacterial Infection. MBio. 2019; 10(3): pii: e00810-19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith AM, McCullers JA: Secondary bacterial infections in influenza virus Infection pathogenesis. Curr Top Microbiol Immunol. 2014; 385: 327–56. PubMed Abstract \| Publisher Full Text\n\nKobzik L: \"ARRIVE Guidelines checklist\". Harvard Dataverse, V1 2019. http://www.doi.org/10.7910/DVN/VQBKLF" }	[ { "id": "56255", "date": "18 Nov 2019", "name": "David H. Dockrell", "expertise": [ "Reviewer Expertise Pathogeneiss of infectious diseases", "especially respiratory tract infections." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nYang et al. explore the potential for of recombinant human gelsolin as a therapeutic intervention in a PR8 H1N1 mouse flu model. They use CD1 mice and use a variety of doses and dosing schedules with a pooled statistical analysis to infer benefits across a range of groups despite some variability in results. Benefits were seen in terms of survival and morbidity scores but not in terms of weight loss. They went on to perform transcriptional profiling and to detect reductions in inflammatory responses and increases in responses associated with tissue repair.\nStrengths of the study are the investigation of a therapeutic approach for a potential life threatening infection and the preliminary hypothesis generating transcriptomic data set which provide clues to potential mechanisms. A few areas of the text should be addressed to strengthen the main conclusions.\nThe authors have had significant statistical input but some further explanation of the statistical approach and its potential benefits to a study such as this would be useful.\nThe study used CD1 mice which may explain some of the variability. A few further details should be added to the methods. The authors should confirm the reason for just studying males. They should confirm animals and groups were co-housed and, in a study with such large numbers, explain whether there were any majors groupings or time periods used in the study which might have contributed to variability. Were mice bought in in small groups or are the subgroups from predominantly the same larger pool bred in house? Also they should confirm the mice were infected at similar times of day.\nThe authors might also comment on the rationale for using human gelsolin. Some differences in response between human and mouse gelsolin have been detected and the authors might wish to comment on this and their similarities and differences.\nThe authors did not find any major differences in weight. Can the authors comment further on this since this is usually a marker of outcome. In addition the mortality curves appeared to be still showing deaths at the end of the study period. Can the authors confirm that the main biologic affect was a delay in mortality rather than prevention? Clarifying this in the text would resolve any uncertainty relating to the conclusion and title.\nThe transcriptomic data is of interest. Can the authors drill down further and confirm any sub-groupings related to inflammation or tissue repair? In particular is their any evidence of an epithelial protective affect such as enhancement of barrier function or epithelial apoptosis inhibition, since this is potentially one of gelsolins biologic functions. I assume there is no additional data from BAL cytokines, cell counts or histology to back up the transcriptomics but if there are any further data, they would strengthen the conclusions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5240", "date": "21 Feb 2020", "name": "Lester Kobzik", "role": "Author Response", "response": "Response to Reviewer #1: The study used CD1 mice which may explain some of the variability. A few further details should be added to the methods. The authors should confirm the reason for just studying males.They should confirm animals and groups were co-housed and, in a study with such large numbers, explain whether there were any majors groupings or time periods used in the study which might have contributed to variability. Were mice bought in in small groups or are the subgroups from predominantly the same larger pool bred in house? Also they should confirm the mice were infected at similar times of day. To address these points, the following text has been inserted into the methods: Only male mice were used due to budgetary and time limits. All mice arrived and were co-housed 1 week prior to the start of the experiments. Each trial used a separate batch of mice. All infections were done at approximately the same time of day (starting at ~10 AM). The authors might also comment on the rationale for using human gelsolin. Some differences in response between human and mouse gelsolin have been detected and the authors might wish to comment on this and their similarities and differences. To address this comment, the following text has been inserted into the methods: We used human rather than murine gelsolin based on prior demonstrations of function of rhu-pGSN in rodent models and because data with the human gelsolin will facilitate clinical translation efforts. The authors did not find any major differences in weight. Can the authors comment further on this since this is usually a marker of outcome. In addition the mortality curves appeared to be still showing deaths at the end of the study period. Can the authors confirm that the main biologic affect was a delay in mortality rather than prevention? Clarifying this in the text would resolve any uncertainty relating to the conclusion and title. We were also puzzled by the lack of weight differences but have no explanation to offer. A small number of the initial trials followed mice longer than the period reported in the paper. Mice surviving to day 12 continued to live up to day 18-21 in these early trials. However, this was not systematically studied so it is fair to say it is a qualitative impression rather than a quantitative result. Extending the trial length would require additional experimentation. Either a significant delay in mortality or true improved survival would provide the same basis for supporting further studies of rhu-pGSN as a therapeutic worth validating in other models (e.g. ferret). Re: the reviewer’s point about mortality, we now explicitly acknowledge in the Discussion:Mice were only followed for 12 days when euthanasia was performed on surviving mice. Since the survival curves were still potentially declining, the ultimate mortality rate could not be confidently ascertained. However, the time to death at a minimum was prolonged with rhu-pGSN over placebo treatment. The transcriptomic data is of interest. Can the authors drill down further and confirm any sub-groupings related to inflammation or tissue repair? In particular is their any evidence of an epithelial protective affect such as enhancement of barrier function or epithelial apoptosis inhibition, since this is potentially one of gelsolins biologic functions. I assume there is no additional data from BAL cytokines, cell counts or histology to back up the transcriptomics but if there are any further data, they would strengthen the conclusions. We did not find any enrichment for the groupings suggested beyond what we reported in the supplemental data spreadsheet. No additional data from the samples listed by the reviewer is available are available, so this interesting topic can only be addressed in future studies. A comment on treatment changes between the two time points for transcriptome profiling was inserted in the Results section: Per protocol, the rhu-pGSN dose was increased in this experiment on day 7, between the 2 timepoints selected for profiling." } ] }, { "id": "56253", "date": "04 Dec 2019", "name": "David S Fedson", "expertise": [ "Reviewer Expertise I am a retired academic general internist with an interest in how to treat the host response to infection." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nF1000RESEARCH – Submitted by David S. Fedson, MND\n\nYang Z, Bedugnis A, Levinson S, DiNubile M, Stossel T, Li Q, Kobzik L. Delayed administration of recombinant plasma gelsolin improves survival in a murine model of severe influenza. F1000 Research 2019;8:1860.\n\nReviewer’s comments\n\nThis straightforward paper presents the results of a study showing that rhu-pGSN improves survival in a mouse model of influenza. My answers to the mandatory questions above indicate the paper is acceptable and requires no significant modification. The comments below are suggestions and are offered in the hope they might help readers. The authors should feel free to ignore them if they wish.\n\nIntroduction, page 3, left column, paragraph 2 - The pathogenesis of influenza involves a dysregulated and injurious host response that is not wholly inflammatory. Inflammation is involved to be sure, but there is also a degree of immunosuppression. Some of the changes (e.g., pulmonary endothelial dysfunction) can be regarded as separate from inflammation and/or immunosuppression. The authors argue that rhu-pGSN is “an attractive candidate (for treatment) because it dampens excessive and injurious inflammation and augments antimicrobial defences”. Rhu-pGSN probably does much more. (See Becker PM et al. Pulmonary vascular permeability and ischemic injury in gelsolin-deficient mice. Am J Respir Cell Mol Biol 2005;28:478-841 and earlier Kuhne W et al. Disintegration of cytoskeletal structure of actin filaments in energy-depleted endothelial cells. Am J Physiol 1993;265(5 Pt2):H1599-6082.)\n\nPage 3, left column, paragraph 4, lines 1-7 - The first 7 lines could be written more tightly. “One of pGSN’s many functions is to dissolve the actin gels that arise from cellular debris, hence its name. These gels form a biofilm that reduces the ability of cellular and humoral defences to gain access to embedded pathogenic organisms. In response, pGSN accumulates at sites of tissue damage. Interaction with actin reduces pGSN’s binding to …” Also, in line 12, delete “final”. This is not the final comment in this paragraph.\n\nPage 3, right column, paragraph 1, lines 3-4 - A sentence could be added: “… levels decrease and probability of mortality. For this reason, pGSN levels have been considered as potential biomarkers of severity for several acute and chronic diseases,7 In addition, as might be predicted, systemic treatment …” (Reference 7 is a remarkably complete review of published studies of gelsolin and should be read by anyone reading this paper.)\n\nPage 3, right column, paragraph 2, lines 1-2 - These lines could be rewritten: “Regarding the treatment of the severe pneumonia often seen in fatal influenza, administration of rhu-pGSN …”\n\nResults, page 5, right column, paragraph 2, lines 12-15 – These lines could be rewritten: “…The only exception was found in trials 15-19 (Table 1) that tested dose regimens that were initially low (>2 mg rhu-pGSN on days 3 to 5/6, then 5 mg daily through day 11). These trials led to weights (compared to day 0) at the …” By including “(compared to day 0)”, it is not entirely clear whether the weights mentioned in lines 16 and 17 (81.4 and 85 g?) are mean weight differences between day 0 and the end of the study in each of the two groups or the mean weight difference between the two groups at the end of the study. I assume it is the former, but the reader would have to check Extended data to be certain.\n\nPage 5, paragraph 2, line 19 and paragraph 3, line 2 - What is the difference (if any) between Extended data and Underlying data? Should Underlying data really be Extended data?\n\nPage 5, paragraph 3, line 5 – This sentence could be rewritten: “… Comparison of lung samples obtained on day 7 from vehicle-treated and rhu-pGSN-treated mice showed …”\n\n“Page 5, paragraph 3, lines 10-1 – This sentence could be rewritten: “… Among down-regulated genes in the rhu-pGSN-treated group, many cytokine and immune-related genes were prominent, including IL-10, IL-12b, …”\n\nPage 7, left column paragraph 1, line 3 – Do the “main findings” in the analysis of down-regulated genes refer to all down-regulated genes or only the top 50? I assume it is the latter, but this should be made clear to the reader.\n\nPage 7, left column, paragraph one, last four lines (and top two lines in the right column) – This is a very long sentence. It’s worth considering a rewrite: “…In worksheets 2-15 in the spreadsheet in Extended data, we present details of the … “\n\nPage 8, right column, paragraph 1, line 2 – It should be “virus stock”, not “viral stock”.\n\nThe authors should be commended for admitting they can’t explain all of their results.\n\nPage 8, right column, paragraph 1- The authors showed rhu-pGSN treatment offered “only a partial (albeit significant) survival benefit”. Among their suggestions for future studies they mention using larger animals and other strains of influenza virus. They overlook the possibility of combining rhu-pGSN with other drug treatments. After all, they showed only a modest increase in survival (roughly 40% in vehicle-treated and 60% in rhu-pGSN-treated mice, respectively). Combination treatment might offer a greater survival benefit. The authors might also emphasize that studies of the effects of gelsolin and treatment with rhu-pGSN on host responses to several infections suggest rhu-pGSN might used in the syndromic treatment of many different infectious diseases.\n\nReferences\n\nIn addition to the two articles on gelsolin and endothelial dysfunction mentioned earlier, the authors might include their more recently published paper:\n\nSelf WH, Wunderink RG, DiNubile MJ, Stossel TP, Levinson SL, Williams DJ, et al. Low admission plasma gelsolin concentrations identify community-acquired pneumonia patients at high risk for severe outcomes. Clin Infect Dis2019; 69(7):1218-253.\n\nTables and Figures\n\nTable 1. Dividing the table into two sections showing results of treatments started on day 3 and day 6 might make it easier for readers to see the important findings. The two sets of findings could be further subdivided into those showing benefit (YES) or not (NO).\n\nFigure 1 - The legends within panels A, C, E, and G show pGSH above vehicle, while in panels B, D, F, and H the two legends are reversed. It would be better if a consistent style could be used throughout the figure. If the legends are changed, the authors should use rhu-pGSN, which is what they used in their studies (not pGSN).\n\nFigure 2. – It would be helpful to include headings (down-regulated and up-regulated) at the top of the two sets of heat maps.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5241", "date": "21 Feb 2020", "name": "Lester Kobzik", "role": "Author Response", "response": "Response to Reviewer #2: This straightforward paper presents the results of a study showing that rhu-pGSN improves survival in a mouse model of influenza. My answers to the mandatory questions above indicate the paper is acceptable and requires no significant modification. The comments below are suggestions and are offered in the hope they might help readers. The authors should feel free to ignore them if they wish. We are grateful for the many constructive comments and suggestions. As allowed by the reviewer, we have left some comments regarding minor layout and sentence construction issues unanswered. However, for the majority we undertook revisions in the manuscript that are reported below: Page 3, left column, paragraph 4, lines 1-7 - The first 7 lines could be written more tightly. “One of pGSN’s many functions is to dissolve the actin gels that arise from cellular debris, hence its name. These gels form a biofilm that reduces the ability of cellular and humoral defences to gain access to embedded pathogenic organisms. In response, pGSN accumulates at sites of tissue damage. Interaction with actin reduces pGSN’s binding to …” This suggested text has been used in its entirety to replace the prior version. Page 5, paragraph 2, line 19 and paragraph 3, line 2 - What is the difference (if any) between Extended data and Underlying data? Should Underlying data really be Extended data? These are rubrics used by F1000 and we followed editorial guidelines in designated data for one or the other grouping. The links to the data at the end of the paper allow the reader to see these data. We cannot better explain the rationale for ‘underlying vs extended”. Page 5, paragraph 3, line 5 – This sentence could be rewritten: “… Comparison of lung samples obtained on day 7 from vehicle-treated and rhu-pGSN-treated mice showed …”This suggested text has been used in its entirety to replace the prior version. Page 7, left column paragraph 1, line 3 – Do the “main findings” in the analysis of down-regulated genes refer to all down-regulated genes or only the top 50? I assume it is the latter, but this should be made clear to the reader.As suggested, we have clarified this point by changing the text as below to indicate that all the down-regulated genes were used for this analysis: We performed gene enrichment analysis of the full down-regulated gene list Page 8, right column, paragraph 1, line 2 – It should be “virus stock”, not “viral stock”.Fixed as suggested. Page 8, right column, paragraph 1- The authors showed rhu-pGSN treatment offered “only a partial (albeit significant) survival benefit”. Among their suggestions for future studies they mention using larger animals and other strains of influenza virus. They overlook the possibility of combining rhu-pGSN with other drug treatments. After all, they showed only a modest increase in survival (roughly 40% in vehicle-treated and 60% in rhu-pGSN-treated mice, respectively). Combination treatment might offer a greater survival benefit. We have added the sentence below to this section to include this helpful idea. We can also speculate that combination therapy with other agents might offer a greater survival advantage. Unrelated to any specific reviewer comment, we added more detailed mention early in the Discussion of why we did not test rhu-pGSN treatment before day 3: In addition to the impractically of initiating earlier therapy right after infection (as opposed to the onset of severe symptoms) in patients, we did not want to interfere with the immediate immune response to influenza given the detrimental consequences observed in some experimental models." } ] } ]	1	https://f1000research.com/articles/8-1860
https://f1000research.com/articles/8-2133/v1	20 Dec 19	{ "type": "Software Tool Article", "title": "SNPector: SNP inspection tool for diagnosing gene pathogenicity and drug response in a naked sequence", "authors": [ "Peter T. Habib", "Alsamman M. Alsamman", "Sameh E. Hassanein", "Ghada A. Shereif", "Aladdin Hamwieh", "Alsamman M. Alsamman", "Sameh E. Hassanein", "Ghada A. Shereif" ], "abstract": "Due to the ability to diagnose diseases early and evaluate the effectiveness of medicinal drugs, single nucleotide polymorphism (SNP) identification receives significant interest. Detection and diagnosis of genetic variation through skill-less computational tools would help researchers reducing the severity of such health complications and improving well-tailored therapies using discovered and previously known information. We introduce SNPector, which is a standalone SNP inspection software, which can be used to diagnose gene pathogenicity and drug reaction in naked genomic sequences. It identifies and extracts gene-related SNPs, and reports their genomic position, associated phenotype disorder, associated diseases, linkage disequilibrium, in addition to various drug reaction information. SNPector detects and verifies the existence of an SNP in a given DNA sequence based on different clinically relevant SNP databases, such as NCBI ClinVar, AWESOME, and PharmGKB, and generates highly informative visualizations of the recovered information.", "keywords": [ "SNP", "Disease", "Python", "Bioinformatics" ], "content": "Introduction\n\nIn recent years, the number of cases of genetically originated diseases has increased, alarming the world and sparking interest in the development of precision medicine using molecular biomarkers. Single nucleotide polymorphisms (SNPs), the most common genetic difference among individuals, occurs in the human genome. These randomized modifications in DNA bases cause alterations in protein sequence residues of amino acids, thus altering their functions, which leads to different disease conditions in individuals1. Several of these SNPs have been identified as disease-related genetic markers that have been used to recognize genes responsible for particular diseases in humans2.\n\nDistinguishing the evidence and the interpretation of a rich range of markers will be necessary to relate major alterations in the SNPs and to discover their connection in the progression of disease. Clarification of the phenotypic-associative mechanisms for these variations is therefore vital for comprehending the sub-atomic subtleties of disease origin and for developing novel therapeutic methods3,4.\n\nAlthough SNPs may exist in various areas of the gene, such as promoters, introns, 5′-and 3′ UTRs, to date, most research has focused on disease-associated SNPs in coding regions or exons, especially non-synonymous SNPs, which may alter the biochemical ability of encoded proteins. In turn, altering gene promoters impact gene expression by changing transcription, binding transcription factor, methylation of DNA and modifications of histones. As a consequence, changes in gene expression, their impact on disease susceptibility, and drug responses can differ depending on the location of the SNP5–7.\n\nWith the expansion of genetic variants, different software could be used to generate new knowledge to support disease diagnosis and drug response studies and to develop new biomarkers for disease identification and drug customization. In this regard, a number of software applications have been developed in the last few years to classify, prioritize and evaluate the impact of genomic variants. For example, the Ensembl Variant Effect Predictor offers access to a large range of genomic annotations, with a variety of frameworks that answer different needs, with easy setup and evaluation methods8. Similarly, SnpEff categorizes the results of genome sequence variations, annotates variants according to their genomic location and estimates the coding effects. Depending on genome annotation, it is possible to predict coding effects such as non-synonymous or synonymous substitution of amino acids, stop codon gains or losses, start codon gains or losses, or frame changes9.\n\nOn the other hand, another tool, PolyPhen-2, assesses the potential impact of the genetic substitution of amino acids on the basis of physical, evolutionary comparative factors and model structural changes. Based on these profiles, the probability of a missense mutation becoming dangerous is measured on the basis of a combination of all these properties10. Similarly, SIFT calculates whether the substitution of amino acids affects protein activity, based on the homology of sequences and the physical properties of amino acids. It may be used for non-synonymous polymorphisms and laboratory-induced missense mutations that naturally occur, to effectively classify the effects of SNPs as well as other types, including multiple nucleotide polymorphisms11.\n\nMoreover, Phyre2 is a web-based suite of tools for predicting and analysing protein structure, function and mutations. It has sophisticated remote homology identification methods to build 3D models, anticipate ligand binding sites, and evaluate the effect of amino acid variants, e.g. non-synonymous SNPs12. Missense 3D uses the user-provided UniProt ID of the query protein, wild-type residue and substitution and other information to generate PDB residue mapping and predict the substitution effect on the 3D protein structure13.\n\nTo conclude the effect and possible phenotype of SNP, these software and web applications require minimum information such as SNP genomic position, SNP ID, allele form, and/or gene name. Acquiring this information requires using different computational tools, extensive time and some analysis skills. Most of the time, only gene sequences are available in which the SNPs are hidden without any additional information.\n\nWe therefore introduce SNPector, a standalone SNP inspection software that can be used to diagnose gene pathogenicity and drug reaction in naked genomic sequences. SNPector identifies and extracts gene-related SNPs, and reports their genomic position, associated phenotype disorder, associated diseases, linkage disequilibrium, in addition to various drug reaction information. It detects and verifies the existence of an SNP in a given DNA sequence based on different clinically relevant SNP databases, such as NCBI ClinVar14, AWESOME15, and PharmGKB16. Lastly, it connects identified SNPs, related diseases and drugs, and produces numerous visualization figures to explain these relationships with the support of different Python modules.\n\n\nMethods\n\nThe SNPector Python tool uses many packages to inspect the existence of SNPs in a given sequence. Moreover, SNPector provides users with detailed visualization figures, highlighting other SNPs with similar mutation effects on protein phosphorylation, ubiquitination, methylation, or sumoylation sites, and predicts substrates of N-acetyltransferase.\n\nAdditionally, SNPector provides the ability to visualize obtained information about the linkage disequilibrium of detected SNPs using various Python packages, such as Matplotlib17, generating a number of figures that summarize vast amounts of previously published data indicating SNPs allelic segregation, association, minor allele frequency. Figure 3 shows an example of illustrations that can be generated through SNPector.\n\nSNPector requires at least Python 3.5, 16GB RAM, i7 cores, and 8 MB.\n\nSNPector was written using Pythpn3 programming language as a standalone package and can be run on different operating systems platforms supported with Python 3.x compilers. To achieve user-friendly usage, the SNPector only accepts input from FASTA sequence (Figure 1) and can be operated from a console through simple command line (Figure 2).\n\n(A) Circos illustration where other SNPs that have same proprieties are located. (B) Lollipop plot shows values by vertical columns (C) Counter Plot between two values creating a different coloured shade in which more contrast means higher value. (D) Numerical schematic showing the distribution between four values by plotting and scaling colour contrast according to other to values. (E) Heat map between SNP linkage disequilibrium matrix to show how two SNPs are linked. (F) Marginal plot combining column graph and plot, both showing the relationship between two values. (G) Dendrogram with heat-map showing how all SNP are linked to each other. (H) Histogram with box plot to compare visually between two values. (I) Plot illustrating the regression fit of two plotted value. (J) 3D plot of three values. (K) Annotated heat-map showing the plotted value.\n\nSNPector uses different SNP record information collected from NCBI ClinVar (159,184 records), AWESOME (1,080,551 records), and PharmGKB18 (3,932 records). Ldlink is an online tool that can be used to assess linkage imbalance (LD) throughout ancestral populations and is a popular method of exploring population-specific genetic framework and functionally navigation disease susceptibility areas19. In SNPector, an Application Program Interface (API) has been programmed to download an LDhap file containing linkage disequilibrium statistics and potentially functional variants for a query variant resulting from the inputted FASTA sequence.\n\nSNPector starts by running BLAST20 software locally to find out the genomic location of a given DNA sequence on human genome. If successful, it retrieves the SNP records located within the query genomic range using NCBI ClinVar. According to retrieved records from the database, the detected SNPs in user-provided queries are marked as wild or mutated. Additionally, more information regarding detecting SNPs records will be retrieved from different implemented databases. This information will be used to generate different illustration figures.\n\nIf the process is successfully finished, SNPector will generate four different files: (A) Text file containing the output BLAST result, where the genomic location of the user-defined sequences is predicted; (B) tab delimited file containing SNPs retrieved by NCBI ClinVar located in the same regions; (C) two files regarding specific SNPs information retrieved from AWESOME and PharmGKB databases; (D) different figures depicting SNPs with a similar mutation effect to the detected SNPs located on other genomic regions, SNP linkage disequilibrium, the relationship between SNP, drug, and phenotype (Figure 3).\n\nTo achive maximum user-freindly usage, SNPector can be run and controlled by command line. SNPector command line structure (Figure 2) is as follows: A) Python3 compiler; B) scan_dna.py: program main script that contain all functions; C) -blaston / -blastoff: in order to initiate BLAST process to provide sequence alignment against the genome to locate where the sequence is situated, if the -blastoff is chosen it will use previous BLAST results; D) -modescan: to scan the given sequence and find out whether SNP occurs or exists in sequence or not, and -modesearch: to extract all SNPs occur in this range of sequence regardless they are exist or not; E) -circoson: draws a Circos figure to illustrate where SNPs with same properties/effect are located; F) -networkon: in order to link between SNPs, diseases and drugs and produces network HTML file; G) -download: activates the API to download data for identified SNPs from LDlink database; H) -vis: produces different figures and plots; I) GivenSequence.fasta: Tte user-provided sequence in FASTA file format. Any of the previous parameters can be deactivated when replaced with -off.\n\n\nUse case\n\nIn this section we provide an example on how to use SNPector to extract SNPs from a naked given sequence without a reference sequence and how these extracted SNPs are linked to disease development and how they affect drug response. We show how to define the arguments of the SNPector function, interpret the results, and make visualizations.\n\nWe use part of an EGFR gene sequence downloaded from NCBI nucleotide database in FASTA format as shown in Extended data: File 121. The EGFR gene FASTA sequence submitted to NCBI contains SNPs that have a clinical effect involved in disease development, such as breast cancer. SNPector uses: (i) NCBI ClinVar database that describes SNP chromosome, position, ID, reference nucleotide, alternative nucleotide, quality, filter, and information to compare and detect SNPs in EGFR sequence that has clinical complications; (ii) PharmGKB database to investigate the SNP effect on disease development and drug response; (iii) AWESOME database to explore SNP effect on phosphorylation, ubiquitination, methylation, and sumoylation sites; and (iv) Ldlink API database of SNP linkage disequilibrium to find out how detected SNPs are linked to other SNPs.\n\nSNPector uses different libraries to import, read, and read data and results. os library is used to run BLAST bash script:\n\n\n\ntime is used to calculate the time that program.\n\n\n\nre refers to regular expression. This library sorts and splits input data with function re.split().\n\n\n\nitemgetter module is used to sort BLAST data according to identity, mismatch, and p-value.\n\n\n\nFrom sys library we used sys.argv[] to convert script to command line, which can be run and controlled from the terminal.\n\n\n\nThen we import the Scripts package to visualize the data as follows:\n\n\n\n\nSNPector variables\n\nTo sort data between the given sequence, Clinvar, AWESOME, BLAST, and PharmGKB, we implement the SNPector variables. This inherits the built-in function open() and nine variables are created as follows:\n\nPharmGKB: data frame describes variant ID, gene name, type of effect, level of evidence, chemicals used to treat the phenotype, and phenotypes;\n\nBLAST_RESULT: data frame lists BLAST output results of alignment of the given sequence against the human genome;\n\nAwesomeDB: data frame lists SNPs chromosome, location, and properties, such as phosphorylation, ubiquitination, methylation, and sumoylation sites;\n\nNCBIclinVar: data frame of SNPs that has clinical impact and involvement in disease;\n\nSNPinDetails: data frame that lists the detected SNPs that SNPector found in the given FASTA sequence;\n\nSNPinDetailsPharmGKB: data frame that lists detected SNPs and its impact on disease development and drug response;\n\nSNPinDetailsAwesome: data frame that lists the properties of detected SNPs;\n\nBLASTfile: function to open and read BLAST output results;\n\nSeqFile: function to read the input file containing the sequence.\n\nEach imported dataset can be found in Extended data21.\n\nRunBLAST() takes the file path of FASTA sequence and starts to align the sequence against the human genome and writes the results to BLAST_RESULT.txt (Extended data: File 221).\n\n\n\nExtractSNP() reads BLAST_RESULT.txt and sort its with itemgetter() according to the identity, length and p-value, then stores the start and end input given sequence (the query) and subject to use later in the extraction step. It also reads the input FASTA sequence file and stores the sequence variable to use in the comparing extraction step. SNPector provides two inspection modes that can be determined from the terminal, Search and Scan. If mode was “-modesearch”, then SNPector begins to extract all SNPs within the query start and end regardless of their existence in the query. In the mode “-modescan”, SNPector will extract only SNPs that exist in the query\n\nSNPector begins to obtain the alternative nucleotides of SNP through the input sequence and obtains the nucleotides that range from SNP position in ClinVar minus the end position of the subject, to the SNP position in ClinVar minus the end position of the subject plus alternative SNP length to ensure the capture of SNPs from the given sequence and also to detect variants with length more than one nucleotide, finally storing it in the “query_nuc_alt” variable.\n\n\n\nAfter the process of extraction the result saved to: “FromAwesom.tsv” file (Extended data: File 321), in which SNPector list all other SNPs that have the same effect in different sites in proteins; “FromNCBI.tsv” (Extended data: File 421), which is list of the SNPs that SNPector detects in a given sequence and retrieves from NCBI ClinVar Dataset; “FromPharmGKB.tsv” (Extended data: File 521), which lista the effect of SNPs in disease development and drug response.\n\nAPIcommands() imports SNP IDs from “FromNCBI.tsv” and uses Ldlink API to download “LDhap.csv” file (Extended data: File 621), which describes the allele frequency of extracted SNPs, “LDmatrix.csv” file (Extended data: File 721), which shows how far detected SNPs are linked to other SNPs, and a file titles with the SNP id (e.g. rs516316.csv) (Extended data: File 821), which includes additional information, such as minor allele frequency, linkage disequilibrium and distance of other SNPs linked to the detected SNP.\n\n\n\nDrawCircos() uses SNP properties from “FromAwesom.tsv” file (Extended data: File 321) and searches for other SNPs that have the same properties. SNPector then imports pycircos package to draw SNP location on Circos (Figure 3A).\n\n\n\nDrawNetwork() draw network using “FromPharmGKB.tsv” (Extended data: File 521) to get the gene name (e.g. EGFR) and by gene name get all SNP that occur in this gene. Using SNP IDs, SNPector obtains disease names caused by these SNPs, and with the disease name SNPector can extract drugs used in treatment for this disease. Finally, with the drug name SNPector can obtain the clinical annotation of the drug. SNPector uses webweb package to draw the network and export it to .html file (Extended data: File 10).\n\n\n\nVisualization() uses data downloaded in the “LDmatrix.csv” file (Extended data: File 721), and the SNP ID file (e.g. rs516316.csv) (Extended data: File 821) to draw other figures (Figures 3B–K).\n\n\nDiscussion\n\nSNPector can collect and retrieve information from the user-provided DNA sequence in the simplest way possible. By integrating different databases into SNPector, it is possible to detect the fluctuations in the abundance of SNPs in query through comparison with known variants of human genome. Such steps are accompanied by the use of online and verified sources to gather previously published details regarding target genomic regions and to generate highly informative visualizations of the recovered information.\n\nMany tools, however, provide SNPs annotation, but they are still limited to the information provided (Table 1). SNPector, on the other hand, provides a new technique that extracts SNP from a naked sequence with no prior information. In addition, another benefit of SNPector is to annotate the discovered SNPs from information retrieved from various known databases.\n\n\nConclusion\n\nOne of the currently growing medical research paradigms is the diagnosis of genetic virulence that accumulates in our genome causing catastrophic health problems. Detection and diagnosis of genetic variation through skill-less computational tools would help researchers reducing the severity of such health complications and improving well-tailored therapies using discovered and previously known information.\n\nSNPector provides and detects all available information about the disease-related SNPs in the given query with minimum user-provided information. It connects between different available information and produces various illustrations depicting SNP related diseases and treatment network, linked disequilibrium, minor allele frequency, similar SNPs with the same mutation effect and other information.\n\n\nSoftware availability\n\nSource code available from GitHub: https://github.com/peterhabib/SNPector\n\nArchived source code as at time of publication: http://doi.org/10.5281/zenodo.355839322.\n\nLicense: MIT\n\n\nData availability\n\nHomo sapiens chromosome 7, GRCh38.p13 Primary Assembly, Accession number NC_000007.14: https://www.ncbi.nlm.nih.gov/nuccore/NC_000007.14?report=fasta&from=55019017&to=55211628\n\nZenodo: SNPector Supplementary Data, http://doi.org/10.5281/zenodo.356979021.\n\nThis project contains the following extended data:\n\n- Supplementary Files 1–10: output files from SNPector for the FASTA sequence use case (NC_000007.14).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors are deeply grateful to Omar S. Abdel-Gaffar, teaching assistant at the College of Biotechnology, Misr University for Science and Technology.\n\nA previous version of this article is available: https://doi.org/10.1101/834580.\n\n\nReferences\n\nChaudhary R, Singh B, Kumar M, et al.: Role of single nucleotide polymorphisms in pharmacogenomics and their association with human diseases. Drug Metab Rev. 2015; 47(3): 281–90. PubMed Abstract \| Publisher Full Text\n\nKong J, Zhu J, Keyser UF: Single molecule based SNP detection using designed DNA carriers and solid-state nanopores. Chem Commun (Camb). 2016; 53(2): 436–9. PubMed Abstract \| Publisher Full Text\n\nWelter D, MacArthur J, Morales J, et al.: The NHGRI GWAS Catalog, a curated resource of SNP-trait associations. Nucleic Acids Res. 2014; 42(Database issue): D1001–D1006. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStranger BE, Stahl EA, Raj T: Progress and promise of genome-wide association studies for human complex trait genetics. Genetics. 2011; 187(2): 367–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchirmer MA, Lüske CM, Roppel S, et al.: Relevance of Sp binding site polymorphism in WWOX for treatment outcome in pancreatic cancer. J Natl Cancer Inst. 2016; 108(5). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFan H, Liu D, Qiu X, et al.: A functional polymorphism in the DNA methyltransferase-3A promoter modifies the susceptibility in gastric cancer but not in esophageal carcinoma. BMC Med. 2010; 8(1): 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRintisch C, Heinig M, Bauerfeind A, et al.: Natural variation of histone modification and its impact on gene expression in the rat genome. Genome Res. 2014; 24(6): 942–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcLaren W, Gil L, Hunt SE, et al.: The ensembl variant effect predictor. Genome Biol. 2016; 17(1): 122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCingolani P, Platts A, Wang le L, et al.: A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin). 2012; 6(2): 80–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdzhubei I, Jordan DM, Sunyaev SR: Predicting functional effect of human missense mutations using PolyPhen-2. Curr Protoc Hum Genet. 2013; 76(1): 7–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNg PC, Henikoff S: SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res. 2003; 31(13): 3812–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKelley LA, Mezulis S, Yates CM, et al.: The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015; 10(6): 845–58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIttisoponpisan S, Islam SA, Khanna T, et al.: Can Predicted Protein 3D Structures Provide Reliable Insights into whether Missense Variants Are Disease Associated? J Mol Biol. 2019; 431(11): 2197–212. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLandrum MJ, Lee JM, Benson M, et al.: ClinVar: public archive of interpretations of clinically relevant variants. Nucleic Acids Res. 2016; 44(D1): D862–D868. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang Y, Peng X, Ying P, et al.: AWESOME: a database of SNPs that affect protein post-translational modifications. Nucleic Acids Res. 2019; 47(D1): D874–D880. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThorn CF, Klein TE, Altman RB: PharmGKB: the pharmacogenomics knowledge base. Methods Mol Biol. In: Pharmacogenomics. Springer. 2013; 1015: 311–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHewett M, Oliver DE, Rubin DL, et al.: PharmGKB: the pharmacogenetics knowledge base. Nucleic Acids Res. 2002; 30(1): 163–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMachiela MJ, Chanock SJ: LDlink: a web-based application for exploring population-specific haplotype structure and linking correlated alleles of possible functional variants. Bioinformatics. 2015; 31(21): 3555–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltschul SF, Madden TL, Schäffer AA, et al.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25(17): 3389–402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHabib PT, Alsamman AM, Hamwieh A: BioAnalyzer: Bioinformatic Software of Routinely Used Tools for Analysis of Genomic Data. Biotechnology. 2019; 10(3): 33–41. Publisher Full Text\n\npeterhabib: peterhabib/SNPector: SNPector: SNP inspection tool for diagnosing gene pathogenicity and drug response in a naked sequence (Version v1.0.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3558393\n\nPeter: SNPector Supplementary Data [Data set]. Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3569790" }	[ { "id": "58286", "date": "08 Jan 2020", "name": "Fakher Rahim", "expertise": [ "Reviewer Expertise Bioinformatics and clinical epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1.\n\nThe rationale for developing the new software tool was not clearly explained. Given lots of closed or similar tools such as SCOPA1, SIFT, PolyPhen-2, and dbSNP, etc., I was expect more adding value of the present study to describe. One important concern associate with such databases is that these tools should get updated regularly with regards to recent GWAS etc. Ideally, these databases are manually curated.\n2.\n\nI have concern about the format of output and interpretation of the results. I think the author should tested this tool even in very small dataset and report that.\n3.\n\nOne major point is that introducing a new tools need some strong evidences to compete with available and known tools. So the authors should compare the validity and SWOT aspects of this tools with the present tools. So, claiming that “SNPector provides and detects all available information” without a good and clear comparison is only lead to adding a tool to the previous sets of tools.\n\nIs the rationale for developing the new software tool clearly explained? No\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? No\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No", "responses": [ { "c_id": "5146", "date": "20 Feb 2020", "name": "Peter Habib", "role": "Author Response", "response": "First of all, we thank the reviewer for his insightful comments/suggestions that have improved the quality of the manuscript. 1. We agree with the reviewer that the rationale for developing the new software tool was not clearly explained so, we edited the introduction and abstract to clarify the goal of SNPector. our idea was to extract the SNPs from a given sequence in FASTA format without the need to do several steps to extract those SNPs and visiting different databases to get the information of detected SNPs. with previous tools, you have to extract the SNPs by aligning against the genome, exporting those detected SNPs in the proper format which is compatible with other tools you need to study SNP effect, migrate the results from database to another to collect all allele frequency and linkage disequilibrium of each SNP, and take the matrices of linkage disequilibrium and excel sheets of allelic frequency and call different scripts and software to visualize the results. in SNPector, with only one command line SNPs extracted, Retrieve information related to drug response and disease development, Collect information of SNP structural effect of different protein critical sites (e.g. phosphorylation sites), downloading linkage disequilibrium and allelic frequency of detected SNP and other linked SNPs on the same chromosome, sorting the data in excel sheets, and finally visualize the downloaded data to be more understandable. 2. We already tested SNPector and the example provided in the paper is itself the testing. we downloaded the part of the EGFR gene, run SNPector, and the results were provided in the paper.3. We agree with the reviewer so, we edited the discussion section and include comparing examples." } ] } ]	1	https://f1000research.com/articles/8-2133
https://f1000research.com/articles/9-128/v1	20 Feb 20	{ "type": "Brief Report", "title": "Differences in total iron content at various altitudes of Amazonian Andes soil in Ecuador", "authors": [ "Benito Mendoza", "Nelly Guananga", "Jesus R. Melendez", "Daniel A. Lowy", "Benito Mendoza", "Jesus R. Melendez", "Daniel A. Lowy" ], "abstract": "Although iron is not contained by chlorophyll, it is indispensable for plants as it plays an essential role in the biosynthesis of chlorophyll. It is a component of many important plant enzyme systems, e.g. cytochrome oxidase, which is responsible for electron transport. Therefore, examining iron content of soils, particularly ionic forms of iron (Fe2+ and Fe3+) is important for fruit growers. In this article, we disclose the total iron content determined in soils (Hyperalic Alisol soil) at three altitudes of Amazonian rainforest in Ecuador. We examine how different altitudes impact the pH and total iron content in the selected study area. We found that total iron content significantly decreases (R2=0.966) at lower altitudes. For future studies, the authors recommend that along with Fe ion content one should determine calcium, microbial biomass, and microbial activity to better understand iron mobility and dynamics of iron uptake in the area.", "keywords": [ "Iron", "total iron in soil", "Hyperalic Alisol", "Amazonian rainforest", "Ecuador" ], "content": "Introduction\n\nTotal iron concentration of soils mainly depends on pH (Colombo et al., 2014; Jelic et al., 2010) and moisture content; and is also affected by root respiration, soil microbial activity, leaching, and erosion (Spectrum Analytic, Inc. 2020). Given that iron deficiency is a regular problem for various crops, it is essential to determine the total iron content of soils (Mengel et al., 2001), particularly in orchards (Simon & Szilágyi, 2003).\n\nIn a highly cited review paper, Bünemann et al., (2018) identify the most frequently used soil quality indicators under agricultural land use: organic matter, pH, available phosphate, and water storage. Soil quality evaluation should specify targeted soil threats, functions, and ecosystem services. The authors of the review recommend developing increasingly interactive assessment tools.\n\nRecently, several studies have been undertaken on the effects on soil quality exerted by various minerals contained in the soil, such as ammonium lactate-soluble potassium and phosphorus content (Jakab, 2020; Li et al., 2020). Also investigated was the impact of various soil cultivation methods on some microbial soil properties (Beni et al., 2017; Sándor et al., 2020; Sándor, 2020; Veres et al., 2015).\n\nIn this article, we report the variations with altitude of the total iron content measured in intact soil in the Amazonian rainforest (in an uncultivated and uninhabited area). Considering that orchards are the most sensitive to iron deficiency, our results are aimed to support local farmers, when they select new areas for fruit plantations. An intact area was chosen as the control for soil samples, which will serve as the reference for future studies initiated in the nearby agricultural region.\n\n\nMethods\n\nA total of 15 soil samples were collected from three altitude levels: 420, 1000, and 1600 m.a.s.l. (meters above sea level) near Tena, Ecuador, on December 10, 2019, from the upper layer (top 20 cm) of Hyperalic Alisol (Ultisols in US Soil Taxonomy) soil (Table 1).\n\nWe measured pH in distilled water for soil/water ratio of 1:25 (w/w) using a glass electrode (Model Seven2Go Advanced Single-Channel Portable pH Meter, Mettler, Toledo). Soil moisture content was determined gravimetrically; drying the soil samples at 105°C for 24 h and weighing the mass loss. We measured allophane using 10.0 ± 0.5 g soil/water (1:2, w/w), soil/water plus 20. mL 1.0 M NaF, soil/water (1:2.5, w/w) + 25 mL 1.0 M NaF, soil/water (1:2.5, w/w) + 25 mL 0.50 M NaF, as described by Singla et al. (2018).\n\nWe determined total iron (all ionic forms) according to modified Blakemore 1981 method described in Singla et al., 2018. Briefly, 50 mL of ammonium oxalate monohydrate (Spectrum Chemical) (0.20 M, pH 3) was added to 1 gram of soil sample. The mixture was shaken with a Model NB-101M Medium Orbital Shaker (N-Biotek, Inc.) in orbital mode, for 4.5 h at 150 rpms. In total 12 hours later, samples were centrifuged for 15 min at 3500 rpm (using Hermle Z400, Hermle, AG, Germany). Double filtration was performed (Whatman no42 filter). A calibration curve was determined from the extracted solution (oxalate ammonium acid 0.20 M) according to Singla et al. (2018). The solution was measured with a Model 240Z Atomic Absorption Furnace Spectrophotometer (Agilent) at a wavelength of 392 nm and with a slit width of 0.2 nm.\n\nWe applied simple linear regression (Z-test) for statistical analysis, using SPSS (version 26) to reveal possible relevant differences in pH values and total iron content at different altitudes.\n\n\nResults and discussion\n\nExamined soil samples in the chosen area were strongly or moderately acidic, with pH values in the range from pH 4.95 ± 0.05 to pH 5.95 ± 0.05 (Table 1). We did not find any meaningful correlation between altitude and pH values, or between pH and total iron content. Moisture content is the highest at 1600 m.a.s.l. Allophane was detected in all samples, which supports the volcanic nature of the sampling area (Fieldes & Perrot, 1986) (Table 1).\n\nTotal iron content significantly decreases (R2=0.966) at lower altitudes (Figure 1). No significant changes in pH were found, and we can explain this by the following:\n\n(i) vegetation at lower lying areas receive less light, so it absorbs a greater quantity of iron ions; so far, there is no relevant literature data on the effect of light intensity on the iron uptake of plants (Borowski, 2013).\n\n(ii) there is a greater concentration of iron-reducing bacteria in the lower lying areas, which seems to be verified by a prior study (Fiedler et al., 2007). This finding is, however, unusual, because such bacteria are typically present in sea water (Bae et al., 2001) and paddy soils (Singla & Inubushi, 2013), rather than in Hyperalic Alisol soils.\n\nHigh moisture content of the soil and organic matter accumulated on the soil surface can make air circulation difficult, hence, anaerobic conditions can develop in lower lying areas.\n\nOur results (from 400 m.a.s.l. to 1000 m.a.s.l.) are comparable with a prior study performed in the same region (Singla et al., 2018), in which the authors report a decrease in iron content for lower laying areas. The main difference between our assessment relative Singla and colleagues’ results is that they observed a radical decrease in iron content above 1000 m.a.s.l., while we found greater iron concentrations at this altitude. Our results are comparable in magnitude to other study findings (Fageria & Stone, 2008) carried out in South American Hyperalic Alisol soils in which high iron content was found at depths of 0–20 cm.\n\n\nConclusions\n\nTotal iron content significantly decreases (R2=0.966) at lower altitudes. Genomics studies could detect possible iron consuming bacterial strains. For future studies, we recommend that in addition to Fe2+ and Fe3+ content one should determine calcium, microbial biomass, and microbial activity. Altogether, this approach would enable a better understanding of iron mobility and dynamics of iron uptake in the area.\n\n\nData availability\n\nFigshare: Raw data for \"Differences in total iron content at various altitudes of Amazonian Andes soil in Ecuador\", https://doi.org/10.6084/m9.figshare.11833554.v2 (Guananga, 2020).", "appendix": "References\n\nBae SL, Kwak K, Kim S, et al.: Isolation and characterization of CO2-fixing hydrogen-oxidizing marine bacteria. J Biosci Bioeng. 2001; 91(5) : 442–8. PubMed Abstract \| Publisher Full Text\n\nBeni Á, Lajtha K, Kozma J, et al.: Application of a Stir Bar Sorptive Extraction sample preparation method with HPLC for soil fungal biomass determination in soils from a detrital manipulation study. J Microbiol Methods. 2017; 136: 1–5. PubMed Abstract \| Publisher Full Text\n\nBorowski E: Uptake and transport of iron ions (Fe+2, Fe+3) supplied to roots or leaves in spinach (Spinacia oleracea L.) plants growing under different light conditions. Acta Agrobotanica. 2013; 66(2): 45–52. Publisher Full Text\n\nBünemann EK, Bongiorno G, Bai Zh, et al.: Soil quality - A critical review. Soil Biology and Biochemistry. 2018; 120: 105–125. Publisher Full Text\n\nColombo C, Palumbo G, He J, et al.: Review on iron availability in soil: interaction of Fe minerals, plants, and microbes. J Soils Sediments. 2014; 14(3): 538–548. Publisher Full Text\n\nFageria NK, Stone LF: Micronutrient Deficiency Problems in South America. Micronutrient Deficiencies in Global Crop Production. 2008; 245–266. Publisher Full Text\n\nFiedler S, Vepraskas MJ, Richardson JL: Soil redox potential: importance, field measurements, and observations. Adv Agron. 2007; 94: 1–57. Publisher Full Text\n\nFieldes M, Perrot KW: The nature of allop-hone in soils. Part III: Rapid field and laboratorytest for allophone. New Zealand Journal of Science. 1986; 9(3).\n\nguananga: Raw data for \"Differences in total iron content at various altitudes of Amazonian Andes soil in Ecuador\". figshare. Dataset.2020. http://www.doi.org/10.6084/m9.figshare.11833554.v2\n\nJakab A: The ammonium lactate soluble potassium and phosphorus content of the soils of north-east Hungary region: a quantifying study. DRC Sustainable Future. 2020; 1(1): 7–13. Publisher Full Text\n\nJelic MZ, Milivojevic JZ, Trifunovic SR, et al.: Distribution and forms of iron in the vertisols of Serbia. J Serb Chem Soc. 2010; 76(5): 781–794. Publisher Full Text\n\nLi S, Xu J, Tang S, et al.: A meta-analysis of carbon, nitrogen and phosphorus change in response to conversion of grassland to agricultural land. Geoderma. Elsevier. 2020; 363: 114149. Publisher Full Text\n\nMengel K, Kirkby E, Kosegarten H, et al.: Iron. In : Mengel K. and Kirkby E.A. (Eds.) Mineral nutrition, 5th edn. Kluwer Academic Publishers, Dordrecht 2001; 553–571.\n\nSándor Zs: Authors’ correction for “Effect of various soil cultivation methods on some microbial soil properties”. DRC Sustainable Future. 2020; 1(1): 21–22. Publisher Full Text\n\nSándor Zs, Tállai M, Kincses I, et al.: Effect of various soil cultivation methods on some microbial soil properties. DRC Sustainable Future. 2020; 1(1): 14–20. Publisher Full Text\n\nSimon L, Szilágyi M, Eds: Mikroelemek a táplálékláncban (Microelements in the food chain). Bessenyei György Kiadó, Nyíregyháza. 2003.\n\nSingla A, Bautista G, Mátyás B, et al.: Altitudinal variations in H and Al ions interchange along with Fe content in Amazonian rainforest soil. La Granja: Revista de Ciencias de la Vida. 2018; 28(2): 42–50. Publisher Full Text\n\nSingla A, Inubushi K: CO2, CH4 and N2O production potential of paddy soil after biogas byproducts application under waterlogged condition. Int J Agric Environ Biotech. 2013; 6(2): 233–239. Reference Source\n\nSpectrum Analytic, Inc.: Iron (Fe++) - Deficiency Symptoms and Using Iron in a Fertility Program. 2020. Reference Source\n\nVeres Z, Kotroczó Z, Fekete I, et al.: Soil extracellular enzyme activities are sensitive indicators of detrital inputs and carbon availability. Applied Soil Ecology. 2015; 92: 18–23. Publisher Full Text" }	[ { "id": "60361", "date": "24 Feb 2020", "name": "Zsolt Kotroczo", "expertise": [ "Reviewer Expertise Soil biology", "soil chemistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComments and suggestions Authors: Benito Mendoza, Nelly Guananga, Jesus R. Melendez; Daniel A. Lowy Title: Differences in total iron content at various altitudes of Amazonian Andes soil in Ecuador\nSummary of the manuscript: The work contains many novelties. The objectives are well founded but I miss some well-formulated hypotheses. This is very important in such well-established and well-planned research. The topic chosen increases the significance of the research. The manuscript is the original research paper of the authors. The subject of the manuscript is interesting and current.\n\nThe theme of the manuscript’s scope fits the aims of the journal. Most probably the manuscript will be of great interest, contributing to the reading of the journal. It is suitable for indexing in F1000Research journal. The basic idea of the experiment was carried out precisely, the study provided large amount of high quality data.\n\nThe Abstract is quite concise and sums up the essence of the manuscript appropriately. The abstract properly introduces the topic, summarizes the methods used and the results obtained. It is summarizes the essence of the manuscript.\n\nKeywords: I suggest replacing some of the key words. The keywords that are included in the manuscript title should be replaced.\n\nIntroduction The chapter is well structured. It fits in well with the manuscript theme, and establishes and complements the whole research topic. This chapter comprehensively supports the whole manuscript with literature. Appropriate and timely references are built in the introduction chapter. The chapter detailed and well processed.\nMethods The methods chapter are appropriate and sufficiently detailed. In my opinion, soil sampling and soil analysis methods are sufficiently detailed, but the research area description is inadequate. For better identification and understanding of the results, I suggest a brief description of the sampling area.\n\nResults The interpretation of results is generally proper. Results chapter is detailed. These chapters are well-structured and properly constructed. This chapters provide detailed and perfectly summarizes the new and novel results. It contains a number of useful experience and findings. The authors show the results using a graph. Evaluation of these results is appropriate and draws realistic conclusions. On the other hand, it make useful and interesting findings that may be interested.\nConclusions The chapter summarizes and well sums up the essence of the manuscript. This chapter explain and justified by the data. I think that the allegations are supported by the data and results.\n\nThe manuscript is suitable for indexing after an above-mentioned minor revisions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "60360", "date": "28 Feb 2020", "name": "Ankit Singla", "expertise": [ "Reviewer Expertise Soil Microbiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary of the content: It should be mentioned that Kotroczo and co-workers present novel research findings considering that currently there is no iron concentration-related study published in the literature in the sampling area that examines differences in soil iron ion concentrations at different altitudional levels. As mentioned in the manuscript, this is especially useful for orchard-growers. The sampling area was well selected because of its specific location, as it is an inherit area, that is close to cultivated lands. The presented results may serve as control results for further agri- and/or horticultural studies. Also unexpected findings about the iron decrease may awaken researchers´ attention, and/or inspire a more complex research (i.e. genomic studies) in the examination area.\n\nAbstract: abstract is well written and informative.\nKeywords: I agree with Reviewer 1 Dr. Zsolt Kotroczo that Keywords should not include those words that also could be found in the title. Please consider to replace the following keywords: Iron, total iron, Ecuador, providing higher possibility for search engines to find the article.\nIntroduction: One of the most challenged part of the evaluation process in case of Brief Report or Short Communications is the Introduction. Despite it is short, I believe that it consists relevant, and up-to-date research findings of the topic (mainly 2019-2020 content).\nMethods: Methods are well written, well referenced. I agree with Reviewer 1 about it would be more fortunate to provide detailed information about the sampling area. However, it is required to clarify soil/water ratio for pH measurement was 1:25 or 1:2.5? Please check first line of determination of soil properties in the methods section.\nResults and Discussion: Brief and straightforward. Consists of statistical results, supporting data is available as well.\nConclusions: Summarizes and concludes from research findings correctly.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-128
https://f1000research.com/articles/8-1852/v1	05 Nov 19	{ "type": "Brief Report", "title": "Reporting of academic degrees in high-impact medical journals", "authors": [ "Nikola Stankovic", "Jerry P. Nolan", "Lars W. Andersen", "Nikola Stankovic", "Jerry P. Nolan" ], "abstract": "Academic degrees following author names are often included in medical research papers. However, it remains unclear how many journals choose to include academic degrees and whether this is more common in certain types of journals. We examined the 100 highest impact medical journals and found that only 24 medical journals reported academic degrees. Moreover, this was substantially more common in journals based in North America compared with Europe. Further research is required to explore the implications of listing academic degrees on the readers’ attitude towards research quality.", "keywords": [ "Academic", "degree", "impact factor", "journals", "publication", "high-impact" ], "content": "Introduction\n\nDuring submission of research papers to medical journals, authors are often asked to include academic degrees, affiliations, and/or job titles. However, journals differ in how they present this information to readers. While some journals include academic degrees following author names, others choose not to list this information on the title page. It is unclear how many journals choose to include academic degrees and whether this is more common in certain types of journals. Among the most influential medical journals, we examined journal factors associated with the inclusion of academic degrees on the title page.\n\n\nMethods\n\nWe identified the hundred highest impact medical journals based on impact factor reported in the Journal Citation Reports1 published in 2018. Characteristics of each journal in regard to specialty, impact factor, primary journal focus, continent, and open access policy were obtained. Data were collected on the presence of academic degrees following author names in the title page by assessment of multiple original research articles from each journal. Approximately ten articles published in July 2018 and August 2019 were assessed for each journal. If there was any discrepancy between the print and the online version, the print version was used. There were no discrepancies within journals for the two time periods.\n\nDescriptive statistics were used to characterize the journals. Categorical data were compared with the Fisher’s Exact Test and continuous data were compared with the Wilcoxon Rank-Sum Test. The association between journal characteristics and the reporting of academic degrees were estimated using multivariable logistic regression.\n\nStatistical analyses were performed in SAS (version 9.4). A two-tailed p < 0.05 was considered significant.\n\n\nResults\n\nOf the 100 highest impact medical journals, 24 journals reported academic degrees on the title page (Table 1). We found that 49% of journals were published in Europe and 51% were published in North America. Only 8% of European journals reported academic degrees while 39% of North American journals reported academic degrees. The median impact factor of journals reporting and not reporting academic degrees was 12 (IQR 11-20) and 15 (IQR 10 – 19), respectfully.\n\naNot able to be estimated as no journal in this category reported academic degrees\n\nbMedian with quartiles\n\nMultivariable analysis showed that North America and a clinical journal focus was associated with increased odds of reporting academic degrees (Table 1). No association was found for the other journal characteristics.\n\n\nDiscussion\n\nAmong the 100 highest impact medical journals, only 24 journals reported academic degrees following author names on the title page. Reporting of academic degrees was substantially more common in journals based in North America compared with Europe.\n\nListing author academic degrees is an editorial policy decision but there is little guidance from the International Committee of Medical Journals Editors (ICMJE) or the American Medical Association (AMA) Manual of Style. Specifically, the ICMJE states “Each author's highest academic degrees should be listed, although some journals do not publish these2.” and the AMA Manual of Style writes “Journals should establish their own policies on the inclusion of authors' degrees.”3. Neither provides a rationale for providing academic degrees and it remains unclear why some journals do and others do not. The marked difference between journals published in North America compared with Europe cannot be explained by the current study but may be a reflection of cultural differences in attitude towards degrees and titles. Further research is needed to explore the implications of listing academic degrees on the readers’ attitude towards research quality.\n\nLimitations of the current study include that we only evaluated high-impact medical journals. Furthermore, we only assessed some journal characteristics. We evaluated only recent issues of these journals and are therefore unable to comment on trends in the use of author academic degrees.\n\nIn conclusion, we found that academic degrees are reported in about one fourth of medical journals and that this practice is more common in North America.\n\n\nData availability\n\nHarvard Dataverse: Replication Data for: Reporting of academic degrees in high-impact medical journals, https://doi.org/10.7910/DVN/KTWS6C4\n\nThis project contains the following underlying data:\n\n- CSV file with the titles of the medical journals investigated\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nThe Journal Citation Report from Clarivate Analytics can only be accessed through an individual or institutional account.", "appendix": "Footnotes\n\n1https://jcr.clarivate.com/JCRLandingPageAction.action Accessed September 7th 2019.\n\n2http://www.icmje.org/recommendations/browse/manuscript-preparation/preparing-for-submission.html#a\n\n3https://www.amamanualofstyle.com/view/10.1093/jama/9780195176339.001.0001/med-9780195176339-div2-10\n\n4Stankovic, Nikola, 2019, \"Replication Data for: Reporting of academic degrees in high-impact medical journals\", https://doi.org/10.7910/DVN/KTWS6C, Harvard Dataverse, V1" }	[ { "id": "56195", "date": "08 Nov 2019", "name": "Saif Aldeen Alryalat", "expertise": [ "Reviewer Expertise Medicine", "bibliometrics", "ophthalmology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nStankovic et al assessed the factors associated with mentioning academic degrees on the journal’s title page. The current study is interesting and sheds a light on an important topic, although the authors chose a restricted sample to analyze (i.e. high impact medical journals). I believe that the current report can be improved by clarifying some points within its methods section, as follows.\n\nIn the methods section, the authors did not define “academic degrees”. In the data associated with this study, they included columns on “Academic degrees”, “job titles”, and “fellowship designations”. In medicine, reporting of fellowships might be more important than the academic degree, especially if the researcher is a clinician working in a non-academic institution. The authors should clearly define what they mean by “academic degree” that they used in their analysis.\n\nIn the methods section, the authors categorized journals according to specialty as general or specific. The authors should detail the basis of this classification. Moreover, the authors categorized open access status into yes, no, or partly, where “partly” is not a common word used to describe the open access status (examples of common, well-defined words include green open access, hybrid access and so on). By going back to the data, I observed several non-open access journals categorized as “partly”, so I would suggest adopting strict criteria for categorization, such as the one suggested by Scopus: “Open Access Journals are indicated as Open Access if the journal is listed in the Directory of Open Access Journals (DOAJ) and/or the Directory of Open Access Scholarly Resources (ROAD)”.\n\nIn the discussion section, the authors should include studies that discussed the impact of an academic degree in publishing, which would complement the points raised by the authors in the present report.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5079", "date": "12 Dec 2019", "name": "Lars Andersen", "role": "Author Response", "response": "Comment 1: “In the methods section, the authors did not define “academic degrees”. In the data associated with this study, they included columns on “Academic degrees”, “job titles”, and “fellowship designations”. In medicine, reporting of fellowships might be more important than the academic degree, especially if the researcher is a clinician working in a non-academic institution. The authors should clearly define what they mean by “academic degree” that they used in their analysis.” Response 1: Thank you for this comment, which we agree will add clarification on the definition of “academic degrees”. Since “fellowship designations” were reported in six medical journals only - which all included academic degrees – we decided not to include a separate analysis for “fellowship designations”. Job titles were present in one medical journal, and this was available online only, why we chose to exclude this category from the final analysis. Thus, we included following clarification on the definition of academic degrees in the methods section of the manuscript: “The presence of academic degrees was defined as the inclusion of academic degree abbreviations following author names (e.g. MD, PhD, RN, MPH etc.).” Comment 2: “In the methods section, the authors categorized journals according to specialty as general or specific. The authors should detail the basis of this classification. Response 2: Agree. The medical journals were categorized as “general”, “specific” or “surgical” based on the specialty heterogeneity reported in the journal. Journals with high heterogeneity of reported specialties were categorized as “general”. In the statistical analysis, “surgical” was grouped with “specific”, since only two journals were categorized as “surgical”. However, to avoid equivocality, we have redefined the basis of our specialty classification for the journals. The journals are now classified according to subject area designation in Scopus. Consequently, the results have been adjusted according to this classification. Thus, we included following phrase in the methods section: “Journals were categorized as “general” or “specific” based on subject area designation in Scopus2, i.e. whether content was primarily related to one specialty (e.g. cardiology) or multiple specialties.” Comment 3 “Moreover, the authors categorized open access status into yes, no, or partly, where “partly” is not a common word used to describe the open access status (examples of common, well-defined words include green open access, hybrid access and so on). By going back to the data, I observed several non-open access journals categorized as “partly”, so I would suggest adopting strict criteria for categorization, such as the one suggested by Scopus: “Open Access Journals are indicated as Open Access if the journal is listed in the Directory of Open Access Journals (DOAJ) and/or the Directory of Open Access Scholarly Resources (ROAD)”.” Response 3: Thank you for this insightful suggestion. We agree with the need to adopt strict criteria for categorization (such as Scopus). We have adopted Scopus’ definition of open access. The journals within the dataset have been reassessed according to Scopus’ definition of open access, and the current dataset, results and manuscript reflect the changes accordingly. Consequently, we have included following phrase in the methods section: “Open access was identified based on open access designation in Scopus2. Scopus registers open access as follows: “In Scopus, journals are registered as being OA journals only if they are registered as Gold OA or Subsidized OA at one or both of the following sources: Directory of Open Access Journals (DOAJ) and the Directory of Open Access Scholarly Resources (ROAD)”3.” Comment 4: “In the discussion section, the authors should include studies that discussed the impact of an academic degree in publishing, which would complement the points raised by the authors in the present report.” Response 4: Thank you for this comment. We agree that it would be valuable to include studies discussing the impact of an academic degree in publishing. However, to our knowledge, no studies have evaluated the impact of reporting academic degrees in journals, why it was not possible to include this in the discussion section. A statement has been provided in the Discussion section to address the lack of studies examining the impact of reporting academic degrees in publishing: “To our knowledge, no studies exist on the impact of reporting academic degrees in medical journals, why further research is needed to explore the implications of listing academic degrees on the readers’ attitude towards research quality.”" } ] }, { "id": "56491", "date": "18 Nov 2019", "name": "Ana Marusic", "expertise": [ "Reviewer Expertise Journal and peer review research" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article addresses an interesting and intriguing question in publishing - why author's academic degrees are published in journals. Editorial guidelines mention this, but do not provide actual guidance. This research showed that journals from North America and from clinical disciplines are more likely to publish the degrees, which is an interesting finding, at least in high-impact journals. The manuscript is written clearly, as a brief report, and follows a logical order, with appropriate statistical analysis.\nI have two major issues:\nThere is no literature overview on this problem (or a statement that there is no evidence on this issue in literature).\n\nThe description of the selection of articles in the set of 100 journals is vague. It is not clear what was the method for sampling \"approximately ten articles ...\" in the two study years. This needs to be clarified.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5080", "date": "12 Dec 2019", "name": "Lars Andersen", "role": "Author Response", "response": "Comment 1: “There is no literature overview on this problem (or a statement that there is no evidence on this issue in literature).” Response 1: Thank you for raising an important point in the manuscript. To our knowledge – following literature review – there are no studies evaluating the impact of reporting academic degrees following author names in medical journals. We agree that a statement addressing that no evidence on this issue is available, will add valuable content to the manuscript. It should be noted, however, that the current study does not evaluate why journals report academic degrees or whether it has an impact on the readers. In this study, we evaluated whether high-impact medical journals reported academic degrees and whether certain journal factors were associated with reporting academic degrees. Further research is needed to evaluate the impact of reporting academic degrees on readers’ perception of research. The following was rephrased in the Introduction section: “To our knowledge, no evidence is available on how many journals choose to include academic degrees and whether this is more common in certain types of journals.” We chose not to include a statement addressing that no evidence is available on the impact of reporting academic degrees following author names in medical journals in the Introduction section, since it may be out of scope with our aim of the current study. However, an appropriate statement has been provided in the Discussion section: “To our knowledge, no studies exist on the impact of reporting academic degrees in medical journals, why further research is needed to explore the implications of listing academic degrees on the readers’ attitude towards research quality.” Comment 2: “The description of the selection of articles in the set of 100 journals is vague. It is not clear what was the method for sampling \"approximately ten articles ...\" in the two study years. This needs to be clarified.” Response 2: We agree that this definition needs to be clarified. We evaluated numerous original research articles in multiple journal issues to assess any discrepancy in reporting of academic degrees over the time period from July 2018 to August 2019. This would account to approximately 10 research articles. No discrepancy in reporting academic degrees during this time period within each journal was identified. Following was rephrased to clarify this issue in the Methods section: “Multiple articles published throughout July 2018 and August 2019 were assessed for each journal. No discrepancy of reporting academic degrees throughout the time period was identified.”" } ] } ]	1	https://f1000research.com/articles/8-1852
https://f1000research.com/articles/8-159/v1	06 Feb 19	{ "type": "Research Article", "title": "Anti-Mullerian hormone levels in female cancer patients of reproductive age in Indonesia: A cross-sectional study", "authors": [ "Achmad Kemal Harzif", "Budi Wiweko", "Putri Addina", "Kartika Iswaranti", "Melisa Silvia", "Ana Mariana", "Kresna Mutia", "Kanadi Sumapraja", "R Muharam", "Gita Pratama", "Achmad Kemal Harzif", "Putri Addina", "Kartika Iswaranti", "Melisa Silvia", "Ana Mariana", "Kresna Mutia", "Kanadi Sumapraja", "R Muharam", "Gita Pratama" ], "abstract": "Background: Efforts in reproductive preservation for cancer patients have become one of the important aspects of cancer management. In fact, decline in reproductive function is known to occur after exposure to anti-cancer treatments. Measuring anti-Müllerian hormone (AMH) levels is known to be the best parameter in predicting ovarian reserves, which indicates reproductive function. In total, 68% of cancer survivors of reproductive age who underwent anti-cancer treatments suffer from infertility. Meanwhile, ovarian reserves also decrease with increasing age. There is ongoing debate on whether the ovarian reserves of cancer patients could be reduced long before exposure to anti-cancer therapy. Therefore, it is important to know whether ovarian reserves in cancer patients decrease before or after anti-cancer therapy. This can help predict the reproductive function in such cases and the effectiveness of ovarian preservation efforts. Methods: A cross-sectional study was conducted, comparing the AMH levels of 44 female cancer patients of reproductive age before cancer therapy, to 44 non-cancer patients of reproductive age (age matched). The biological ages from both groups were adjusted using the Indonesian Kalkulator of Oocytes. Results: The median age in both groups was 28 years old. The AMH levels in the cancer group were found to be significantly lower in contrast to those in the non-cancer group (1.11 [0.08-4.65] ng/ml vs. 3.99 [1.19- 8.7]; p- value <0.001). Therefore, the biological age in the cancer group was 10 years older than that of the non-cancer group, indicating that ovarian aging occurs earlier in cancer patients. Conclusions: AMH levels of cancer patients of reproductive age were already reduced before cancer therapy, given an older biological age, in contrast to that of the non-cancer patients. Proper counseling and implementation of fertility-preserving methods is highly recommended in this group of patients.", "keywords": [ "Anti Mullerian Hormone (AMH)", "Ovarian Reserve", "Cancer in Reproductive", "Biological Age" ], "content": "Introduction\n\nReproductive preservation efforts for cancer patients have become an important aspect of cancer management. Of all female cancer cases worldwide, 10% of cases occur during reproductive age1. Most cancer patients do not have an opportunity to preserve reproductive function prior to cancer treatment, and decline in reproductive function is known to occur after exposure to cancer treatments, whether it be chemotherapy or radiotherapy. In total, 68% of cancer survivors of reproductive age who have undergone anti-cancer treatments experience infertility, amenorrhea, reduction of ovarian reserves, and premature ovarian failure. Ovarian reserves also decrease with increasing age2,3, therefore, the American Society of Clinical Oncology (ASCO), and the American Society for Reproductive Medicine (ASRM) have recommended that consultation on fertility preservation methods prior to cancer therapy should be provided4. However, there is an ongoing debate on whether the ovarian reserves of cancer patients could be reduced long before exposure to anti-cancer therapy.\n\nThe measurement of the anti-Müllerian hormone (AMH) levels is known to be the best parameter for predicting ovarian reserves, which represent reproductive function5. Low AMH levels reflect a low ovarian reserve6,7.\n\nSeveral studies have shown that in breast cancer and lymphoma patients, ovarian reserves are already decreased prior to cancer therapy. However, other studies have shown that a decreased ovarian reserve is only associated with increasing age. It is, therefore, important to know whether the ovarian reserves in cancer patients decrease before or after therapy. This will help predict the reproductive function in such cases and the effectiveness of ovarian preservation efforts.\n\n\nMethods\n\nThis is a cross-sectional study that compares the AMH levels of reproductive age cancer patients before cancer treatment with non-cancer patients. We enrolled 88 subjects; 44 cancer patients of reproductive age before cancer treatment and 44 non-cancer patients (similar age). For cancer group data, patients were recruited from three places; Obstetrics and Gynecology Polyclinics, Hematology Oncology Polyclinics of Cipto Mangunkusumo Hospital, Obstetrics and Gynecology Polyclinics and Inpatient Unit of Dharmais Cancer Hospital, from May 2015 to December 2017. For non-cancer group data, AMH levels were obtained as secondary data from our previous study8, from which we selected patients with a similar age as patients in the cancer group. Inclusion criteria for this study were (a) Cancer and non-cancer patients aged 17–40 years (b) Cancer patients who have never had a history of cancer therapy: chemotherapy/radiation, de-bulking tumors (specifically gynecology); and exclusion criteria were (a) respondents who not willing to be a participant in this study and (b) incomplete of filling informed consent. The sample size was derived from the formula n=z2pqd2 where n is the minimum sample size, Z is the standard normal deviation, as in majority of studies p values are considered significant below 0.05 hence 1.96 is used in formula, p is expected proportion in population based on previous studies (0.36), q is 1-p (0.64), d is the absolute error or precision (0.1 or 10%). Therefore, the total sample size for this study was calculated to be 88 sample subjects and the comparison of cancer groups before cancer therapy and non cancer group was 1:1 meaning the total sample size for each group was 44 subjects. Unpaired numerical analysis formulas were used to compare AMH levels between cancer patients in reproductive age before cancer therapy with non-cancer patients. The standard deviation for each group are calculated separately, and result shows S1 was standard deviation of the cancer group by: 1,088 ng/dl (mean 1.4 ng/dl) and S2 was standard deviation of non-cancer group by : 2.20 ng/dl (mean 4.37 ng/dl). For calculate the minimum number of samples that can be used in this study, and also know the effect size and power of the study so all collected data were inputted to G Power software version 3.0.10. As a result, minimum sample subject count for each group of at least 7 subjects, with actual P 0.83.\n\nData collection was performed by consecutive sampling. In cancer group participants, they were given an explanation of the purpose and benefits of the study, and as asked to provide written consent to participate in the study. The written approval sheet was signed by the patient and the researcher. Researchers registered cases and filled out research status forms (Supplementary File 1 and Supplementary File 2). Blood serum (3 ml) from the cubital vein was collected from the cancer group, and stored in vacutainer without anticoagulants. This serum then sent to FKUI Integrated Laboratory for AMH measurement using ELISA techniques by AMH GEN II ELISA Beckman Coulter REF A79765 (with VMax machines). ELISA was performed to the manufacturer’s protocol. After obtaining the results of AMH levels, data analysis was performed. Full method for AMH hormone examination for cancer groups were taken 3 ml of venous blood from the cubital vein of the media and stored in a vacutainer without anticoagulants. After liquefaction and at room temperature, the AMH examination was carried out manually with a micro ELISA technique read with a VMAX type kinetic microplate reader with Softmax Pro Software version 5.4.1.\n\nStatistical analyses were performed using IBM SPSS version 20. Data on ovarian reserves expressed in AMH levels from the cancer patients and the non-cancer patients are presented as medians (minimum-maximum) because the data were not normally distributed. Mann-Whitney tests were performed to compare the differences in ovarian reserves (AMH levels) between the cancer group and the non-cancer group.\n\nThe Ethics Committee of the Faculty of Medicine from Universitas Indonesia approved this study on May 5, 2015 (reference number: 286/UN2.F1/ATIK/2015). All prospective subjects received an explanation from the main researcher and additional researchers regarding the procedures for conducting research. The decision to follow or refuse to follow the research was taken by informed consent. All data will be kept confidential and the subject had the right to know all the results of the examination carried out.\n\n\nResults\n\nThe 88 subjects participated in this study, with similar characteristics between the two arms. The mean age for cancer and non-cancer groups was the same, 28 years. For the cancer patient menarche age was 11 years. Body mass index for cancer patients was 20.7 ± 3.89, and non-cancer patients 19.5 ± 2.561. Both groups have the same parity average of 0. The mean AMH level for cancer patients was 1.11 ng/ml (0.08–4.65 ng/ml), and for non-cancer patients 3.99 ng/ml (1.19–8.7 ng/ml). The biological age for cancer patients based on an AMH level of 1.1 ng/ml was 38 years old, and for non-cancer patients 3.99 ng/ml corresponded to 28 years old. Characteristics of research subjects can be seen in Table 1. Of all the cancer patients recruited in the study, 14 subjects were gynecologic cancer patients and 30 subjects were non-gynecologic cancer patients, the distribution of cancer cases can be seen in Table 2.\n\nNotes: *Biological age based on AMH level is in accordance with the Indonesian Calculator of Oocyte. BMI: Body mass index, AMH: Anti-Mullerian hormone\n\nOverall, the AMH levels were abnormally distributed (Kolmogorov-Smirnov test for normality, p-value <0.001) with a median level of AMH 2.06 ng/ml (0.08–8.78 ng/ml). AMH levels in the cancer group were lower than that of the non-cancer group (1.11 [0.08–4.65] ng/ml vs. 3.99 [1.19–8.7]; p-value <0.001). This study also showed that there were no statistically significant differences between the AMH levels of the gynecologic cancer patients 0.965 ng/ml (0.08–2.65 ng/ml) and the non-gynecological cancer patients before treatments 1.49 ng/ml (0.08–4.65 ng/ml); p-value 0.162.\n\n\nDiscussion\n\nIn line with advances in cancer therapy, the survival rate of cancer patients also increases. Unfortunately, this rate of improvement does not coincide with an increased quality of life, especially in regards to reproductive function. Currently, the best parameter to measure ovarian reserves is by measuring serum AMH levels; AMH levels can also help predict biological age, which is of more importance when assessing reproductive function.\n\nThere are several types of cancer known to be associated with decreased ovarian reserves prior to cancer therapy, such as Hodgkin and Non-Hodgkin’s lymphoma, and breast cancer. Lawrenz et al. demonstrated that the AMH levels of lymphoma patients were lower than that in non-cancer patients, with mean AMH levels of 2.06 ng/ml vs. 3.20 ng/dl (p- value <0.05)9. Su et al. conducted a similar study wherein the AMH levels of the breast cancer patients were lower than that of the non-cancer patients, 0.6 ng/ml vs. 1.1 ng/ml (p- value <0.001)10. Van et al. also conducted a broader study in 2014 comparing the AMH levels in young cancer patients (<18 years) before treatment with non-cancer patients (age-matched). The results showed that AMH levels in the cancer group were significantly lower than that of the non-cancer group, 1.4 mg/l vs. 3.0 mg/l (p- value <0.001)11. The results of these three studies were similar to that of our study; the AMH levels of the 44 reproductive age cancer patients prior to cancer treatment were lower than that of the non-cancer patients (1.11 [0.08–4.65] ng/ml vs. 3.99 [1.19–8.7] ng/ml; p- value <0.001).\n\nAlthough some studies have shown that the ovarian reserves of reproductive-age cancer patients before treatment are lower than that of the non-cancer patients, the factors that directly affect AMH levels are still unknown. Several studies have suggested genetic mutations, such as BRCA gene mutations, may affect ovarian reserves in breast cancer patients12–14; the effects of high cytokines in lymphoma patients indirectly affect the ovarian reserves as well15,16. In non-cancer populations, Jung et al. has proven that only the age of menarche affected the concentration of AMH (age of menarche <12 years vs. ≥14 years, 0.90 ng/mL vs. 1.12 ng/mL), while ethnicity, BMI, education level, smoking status, height, and menstrual cycle are not associated with AMH concentrations17. Su et al. found that age, BMI, parity, and smoking status are not associated with decreased levels of AMH in patients with breast cancer10.\n\nIn contrast to other types of non-gynecological cancers, reproductive preservation in gynecological cancers is not widely discussed. This is related to the fact that the genital organs themselves are involved in the cancer and the local treatments of such cancers are destructive to the organs of reproduction18. Therefore, in this study, we compared the AMH levels of gynecologic patients of reproductive age before receiving cancer treatment with the non-gynecological cancer patients, and we found no significant difference between AMH levels (0.965 ng/dl vs. 1.49 ng/dl; p- value 0.162). However, the unequal size of the gynecological and non-gynecological cancers subject groups in our study may have affected the results. With increasing age the capability of a woman to produce appropriate quality oocytes, and quantity of oocytes decreases; this process, called ovarian aging, is defined as a gradual decrease in both the quantity and quality of the oocytes. The decrease in the quality and quantity of these oocytes is related to chronological age and biological age19. Chronological age is determined by the passage of time from birth, while the biological age is determined by physiology20. Biological age affects the reproductive function more than chronological age, besides that ovarian reserve is a good marker for representing the biological age of the ovary. In this study, the biological age of the cancer group was 10 years higher than that of the non-cancer group, indicating that ovarian aging occurs earlier in cancer patients. This would indicate that reproductive preservation methods should be offered long before cancer patients undergo therapy.\n\nThere are many reproductive preservation techniques; however, one of technique for cancer patients is ovarian cryopreservation. This technique consists of two methods: the slow cooling method and vitrification ovary. Based on the research conducted by Wiweko et al, ovarian vitrification techniques are superior to the slow cooling method21. Ovarian vitrification does not alter the morphology of the granulosa cells, or the stromal and ovarian collagen components22. It has also been shown to not change the morphology of the pre-antral follicle nor does it increase the risk of cell apoptosis21,22. Other options that may still be offered to cancer patients of reproductive age are oocyte cryopreservation and embryo cryopreservation. In patients who already have a partner or are married, embryo cryopreservation can be offered, however, this may delay cancer therapy due to the ovarian stimulation process required for this technique. In addition to this, estrogen exposure given at the time of ovarian stimulation may have an adverse effect in estrogen sensitive tumors. Unlike embryo cryopreservation, cryopreservation of the oocytes can be used for single women who can undergo the stimulation cycle, but the effectiveness of this technique is very low. The pregnancy and delivery rates range from 1 to 5% per frozen oocyte23. The current weakness of this study is that the other gynecological abnormalities that may affect AMH levels in both groups were not included in the screening criteria for exclusion, such as genetic factors and the type/stage of cancer or other diseases which may affect the levels of AMH, were also not studied.\n\n\nConclusions\n\nThis study showed that the AMH levels in reproductive age patients before receiving therapy were lower in contrast to that in the non-cancer patients (1.11 ng/ml vs. 3.99 ng/ml; p- value <0.001). In addition to this, there was no significant difference in the AMH levels between the gynecologic and non-gynecologic cancer groups before treatment (0.965 ng/dl vs. 1.49 ng/dl; p- value 0.162).\n\n\nData availability\n\nF1000Research: Dataset 1. All raw data and demographic information obtained from subject during the present study, https://doi.org/10.5256/f1000research.15728.d22918624.", "appendix": "Grant information\n\nThis work is supported by Hibah PITTA 2018 funded by DRPM Universitas Indonesia No.5000/UN2.R3.1/HKP.05.00/2018.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors wish to thank all the staff members from INA-REPROMED, Polyclinic Obstetrics and Gynecology, Division of Hemato-oncology Department of Internal Faculty of Medicine Universitas Indonesia, Departement of Internal Medicine and all the staff members from Kanker Dharmais Hospital, Jakarta. We also would like to thank to the professional copyedited by Editage for helping us improve our manuscript by copyedited and given us a thoughtful comments in this manuscript as well.\n\n\nSupplementary material\n\nSupplementary File 1: Questionnaire and informed consent form in Bahasa Indonesia.\n\nClick here to access the data.\n\nSupplementary File 2: Questionnaire and informed consent form in English.\n\nClick here to access the data.\n\n\nReferences\n\nPatel AA, Mini S, Sutaria RP, et al.: Reproductive health issues in women with cancer. J Oncol Pract. 2008; 4(2): 101–105. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeirow D, Nugent D: The effects of radiotherapy and chemotherapy on female reproduction. Hum Reprod Update. 2001; 7(6): 535–543. PubMed Abstract \| Publisher Full Text\n\nOktem O: Quantitative assessment of the impact of chemotherapy on ovarian follicle reserve and stromal function. Cancer. 2007; 110(10): 2222–2229. PubMed Abstract \| Publisher Full Text\n\nDas M, Shehata F, Moria A, et al.: Ovarian reserve, response to gonadotropins, and oocyte maturity in women with malignancy. Fertil Steril. 2011; 96(1): 122–125. PubMed Abstract \| Publisher Full Text\n\nFong SL, Laven JSE, Cammel HF, et al.: Assessment of ovarian reserve in adult childhood cancer survivors using anti-Müllerian hormone. Hum Reprod. 2009; 24(4): 982–990. PubMed Abstract \| Publisher Full Text\n\nAnderson RA, Wallace WH: Antimüllerian hormone, the assessment of the ovarian reserve, and the reproductive outcome of the young patient with cancer. Fertil Steril. 2013; 99(6): 1469–1475. PubMed Abstract \| Publisher Full Text\n\nvan Rooij IA, Broekmans FJ, te Velde ER, et al.: Serum anti-Müllerian hormone levels: a novel measure of ovarian reserve. Hum Reprod. 2002; 17(12): 3065–3071. PubMed Abstract \| Publisher Full Text\n\nWiweko B, Prawesti DM, Hestiantoro A, et al.: Chronological age vs biological age: an age-related normogram for antral follicle count, FSH and anti-Mullerian hormone. J Assist Reprod Genet. 2013; 30(12): 1563–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLawrenz B, Fehm T, von Wolff M, et al.: Reduced pretreatment ovarian reserve in premenopausal female patients with Hodgkin lymphoma or non-Hodgkin-lymphoma--Evaluation by using antimüllerian hormone and retrieved oocytes. Fertil Steril. 2012; 98(1): 141–144. PubMed Abstract \| Publisher Full Text\n\nSu HI, Flatt SW, Natarajan L, et al.: Impact of breast cancer on anti-mullerian hormone levels in young women. Breast Cancer Res Treat. 2013; 137(2): 571–577. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Dorp W, van den Heuvel-Eibrink MM, de Vries AC, et al.: Decreased serum anti-Müllerian hormone levels in girls with newly diagnosed cancer. Hum Reprod. 2014; 29(2): 337–342. PubMed Abstract \| Publisher Full Text\n\nGiordano S, Garrett-Mayer E, Mittal N, et al.: Association of BRCA1 Mutations with Impaired Ovarian Reserve: Connection Between Infertility and Breast/Ovarian Cancer Risk. J Adolesc Young Adult Oncol. 2016; 5(4): 337–343. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLansdorp PM: Repair of telomeric DNA prior to replicative senescence. Mech Ageing Dev. 2000; 118(1–2): 23–34. PubMed Abstract \| Publisher Full Text\n\nMcPherson JP, Hande MP, Poonepalli A, et al.: A role for Brca1 in chromosome end maintenance. Hum Mol Genet. 2006; 15(6): 831–838. PubMed Abstract \| Publisher Full Text\n\nParadisi R, Vicenti R, Macciocca M, et al.: High cytokine expression and reduced ovarian reserve in patients with Hodgkin lymphoma or non-Hodgkin lymphoma. Fertil Steril. 2016; 106(5): 1176–1182. PubMed Abstract \| Publisher Full Text\n\nFabbri R, Pasquinelli G, Magnani V, et al.: Follicle features in adolescent and young adult women with Hodgkin's disease prior to chemotherapy: a preliminary report. Reprod Biomed Online. 2011; 23(6): 799–805. PubMed Abstract \| Publisher Full Text\n\nJung S, Allen N, Arslan AA, et al.: Demographic, lifestyle, and other factors in relation to antimüllerian hormone levels in mostly late premenopausal women. Fertil Steril. 2017; 107(4): 1012–1022.e2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAubard Y, Piver P, Pech JC, et al.: Ovarian tissue cryopreservation and gynecologic oncology: a review. Eur J Obstet Gynecol Reprod Biol. 2001; 97(1): 5–14. PubMed Abstract \| Publisher Full Text\n\nAlviggi C, Humaidan P, Howles CM, et al.: Biological versus chronological ovarian age: Implications for assisted reproductive technology. Reprod Biol Endocrinol. 2009; 7: 101. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBroekmans FJ, Kwee J, Hendriks DJ, et al.: A systematic review of tests predicting ovarian reserve and IVF outcome. Hum Reprod Update. 2006; 12(6): 685–718. PubMed Abstract \| Publisher Full Text\n\nWiweko B, Andriyana H, Aulia A: Ovarian Tissue Vitrification as a Method for Ovarian Preservation in Women with Cancer: an Analysis of Granulose Cell Apoptosis. Indonesian Journal of Obstetrics and Gynecology (INAJOG). 2016; 4(2): 88–92. Publisher Full Text\n\nWiweko B, Maidarti M, Mansyur E, et al.: Ovarian tissue vitrification as a method for fertility preservation: A study of follicle number and morphology after vitrification IVF Lite. 2014; 1(3): 148–152. Publisher Full Text\n\nChang HJ, Suh CS: Fertility preservation for women with malignancies: current developments of cryopreservation. J Gynecol Oncol. 2008; 19(2): 99–107. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarzif AK, Wiweko B, Addina P, et al.: Dataset 1 in: Anti-Mullerian hormone levels in female cancer patients of reproductive age in Indonesia: A cross-sectional study. F1000Research. 2018. https://www.doi.org/10.5256/f1000research.15728.d229186" }	[ { "id": "48874", "date": "10 Jun 2019", "name": "Nao Suzuki", "expertise": [ "Reviewer Expertise Fertility preservation for the young cancer patients", "oncology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-written manuscript about Anti-Mullerian hormone levels in female cancer patients of reproductive age in Indonesia. The aim of the study is to predict the reproductive function of cancer patients. The major limitation of the study is a low number of participants in two groups. I have a few other comments for the authors.\nPage 3 left panel L 21: I think the AMH levels are known to be one of the best parameters for predicting ovarian reserves. I do not think this is the best parameter. I need the author's opinions. Page 3 left panel L 43: Please add the information about the past history of pregnancy and birth, and infertility treatment in the non-cancer group. Whether there is racial difference? Please add the author's opinions in the discussion. Please add more precise opinions regarding the reasons why ovarian aging occurs earlier in cancer patients.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4703", "date": "12 Jul 2019", "name": "Achmad Kemal", "role": "Author Response", "response": "We are grateful for the helpful feedback by the reviewer that helped us improve the quality of our manuscript. We carefully responded to all points and have modified the manuscript accordingly. Please see below our detailed response to comments. Page 3 left panel L 21: I think the AMH levels are known to be one of the best parameters for predicting ovarian reserves. I do not think this is the best parameter. I need the author's opinions. Answer: Yes, we agree that AMH level is one of the best parameters for predicting ovarian reserves. The correction has been made in the manuscript Page 3 left panel L 43: Please add the information about the past history of pregnancy and birth, and infertility treatment in the non-cancer group. Answer: We have added the information for the inclusion and exclusion criteria. The highlighted sentences are the additional information. “…Inclusion criteria for this study were (a) Cancer and non-cancer patients aged 17–40 years (b) Cancer patients who have never had a history of cancer therapy: chemotherapy/radiation, de-bulking tumors (specifically gynecology) (c) Non-cancer patients who never had a history of chemotherapy/radiation. No prior pregnancy or infertility treatment for the non-cancer group; and exclusion criteria were (a) respondents who not willing to be a participant in this study (b) incomplete of filling informed consent and (c) former cancer patient. The informations have been added to the manuscript. Whether there is racial difference? Please add the author's opinions in the discussion Answer: We thank the reviewer for this comment. As explained in Page 5 left panel L 45, Jung et al found that there was no significant variations in AMH concentrations between Asian women and white women (age-adjusted model ; p < 0.77 , multivariable model ; p < 0.62). Although, Bleil et al. found there was a statistically significant race/ethnicity-by-linear age interaction among healthy and regularly cycling women, indicating that differences in AMH levels between race/ethnic groups varied as a function of age. The differences result between previous studies may arise from the varying environment conditions, and heterogeneous character of the populations studied. Serum AMH levels may be influenced by genetic and environmental factors. Our study found that the decreasing level of AMH in cancer group before therapy was related to older of biological age. The result of biological age from both groups were adjusted by Indonesian Kalkulator of Oocytes (IKO) and referred to previous study from Wiweko et al among Indonesian women who went through AMH level test. By using data from this study as reference, we could exclude racial differences of AMH. Our opinions have been added in the discussion of this manuscript. Please add more precise opinions regarding the reasons why ovarian aging occurs earlier in cancer patients. Answer: There is a hypothesis that reported a decreased number of ovarian follicles and an increased gonadotropin level which caused an inflammatory environment and changing epithelial cell surface and the development of tumors in the ovary. Other studies have suggested genetic mutations were associated with ovarian aging. Johnson et al observed BRCA2 carriers had significantly lower AMH levels compared to healthy, low-risk women and had increased odds of having a low AMH (OR 3.69, 95% 1.34–10.19, p=0.012). BRCA 2 as tumor suppressor genes which involved in the regulation of follicular pool through impairment of DNA repair pathway and affected ovarian reserves in breast cancer patients. A study in lymphoma patients showed significantly lower AMH levels than in the control group. There is a strong negative correlation between AMH with SIL-2R, IL-6, and IL-8 cytokines exists. Van Dorp et al found that a significantly lower serum AMH levels of girls with childhood cancer compared with healthy age-matched controls (standard deviation scores (SDS) 20.8, p<0.001). Potential cause of the decreased AMH independently of childhood cancer subtype might be impaired granulosa cell function due to an impaired DNA repair mechanism. Our opinions have been added to the manuscript. We carefully have modified the manuscript according to the editor instructions. Thank you for your considerations, we looking forward to any helpful feedback. Best Regards, Achmad Kemal" } ] } ]	1	https://f1000research.com/articles/8-159
https://f1000research.com/articles/9-125/v1	19 Feb 20	{ "type": "Case Report", "title": "Case Report: Combined nonsurgical and surgical therapy of dens invaginatus of a microdontic maxillary lateral incisor", "authors": [ "Edmond Koyess" ], "abstract": "This report describes the management of an uncommon case of dens invaginatus of a microdontic upper lateral incisor, with an extended apical lesion. Dens invaginatus is a developmental abnormality of a tooth where enamel and dentin fold into the pulpal space. This abnormal anatomy, and the separation of two distinct root canal spaces, complicates conventional treatment, making the apical portion inaccessible to instrumentation and impeding disinfection of the canal space. The coexistence of dens invaginatus affecting a microdontic tooth is a rare anomaly found in the literature. This case report describes a young female patient with dens invaginatus affecting a microdontic maxillary lateral incisor, combined with necrotic pulp and apical periodontitis. The conventional treatment was completed first to disinfect the coronal portion of the accessible pulpal space. At a subsequent appointment, it was completed by a surgical approach to cleanse and seal the apical part of the root canal space. The tooth was then restored, and the orthodontic treatment was initiated. One-year follow-up demonstrated a complete healing of the apical lesion.", "keywords": [ "Dens invaginatus", "pulpectomy", "apicoectomy" ], "content": "Introduction\n\nTooth development may produce various anomalies during differentiation of sensitive structures, such as the dental papilla. Genetic causes as well as local trauma and microbial encroachment upon immature dental tissues may result in anatomical structures that are difficult to negotiate when endodontic treatment may be required1. Reported prevalence of permanent teeth affected with dens invaginatus (DI) ranges from 0.3% to 10%; after routine examination of individuals, this anomaly was observed in 0.25% to 26.1%2. The most frequently affected tooth in permanent dentition appears among maxillary lateral incisor (47%)3. Upon eruption, remnants of the dental papilla or connective tissues from the periodontium may exist in the invagination. These elements might undergo necrosis and provide a nutrient-rich conditions for bacterial growth due to infiltration from the mouth flora2. The early diagnosis of DI is important as it helps in preventing the occurrence of complications from caries through to pulpal degeneration, and periodontal implications, requiring more advanced endodontic procedures later in life3. Among treatment options for DI, the operator might opt for a restoration of the defect, nonsurgical endodontic or surgical treatment, or extraction3. Morphological changes in the root canal may be associated with the invagination itself1,4. Based on radiographic observation, the depth of invagination and connection with periodontal ligament or periapical tissues, Oehlers classified DI into three categories. The three types are: Type I – small invagination, seen only on a radiograph and affecting the cervical third of the root exclusively; Type II – a more severe condition, advancing toward the pulp chamber and extending toward the middle third of the affected root; Type III – the most severe invagination compromising the remaining apical third of the root5. Endodontic treatment is complicated due to the unusual anatomical presentations of the canal spaces(s), which inhibits proper cleansing and shaping of these spaces6.\n\nMicrodontic teeth with DI are uncommon and present with a reduced labial to lingual and/or mesial to distal diameter. To date, examination of reported cases in literature, revealed the description of only two clinical situations, both affecting maxillary lateral incisors7. Our web search (Table 1), revealed more instances of case reports concerning microdontia combined with DI in peg-lateral maxillary incisors. One described a tooth with three patent canals to the apical foramen; in another case report, the peg-lateral had five canals with the same pathway to the foramen4,8. Treatment modalities varied according to clinical conditions: conventional endodontics alone, conventional endodontics followed by surgical treatment, and conventional endodontics with pulp revascularization. This case reports a Type II dens invaginatus with the formation of two distinct root canal spaces. Each canal was treated separately, and with different approaches: one portion of the canal was conventionally accessible through the crown and was treated non-surgically, while the other canal portion could only be reached surgically, with curettage of the periapical abscess, followed by root end cavity and filling.\n\nMTA: Mineral Trioxide aggregate.\n\n\nCase report\n\nA 16-year-old caucasian female patient presented to our private practice in October 2017 for evaluation and eventual endodontic therapy of a maxillary right lateral incisor (Figure 1). The patient had no significant medical history, and the extra-oral exam revealed no unusual findings. The patient’s chief complaint was pain during mastication and swelling in the apical mucosa of the affected tooth. The patient was currently undergoing orthodontic treatment. The maxillary right lateral incisor exhibited a small crown volume with conical shape. Clinical tests on this tooth revealed tenderness to percussion and palpation, with grade 2 mobility. A negative response resulted from the thermal (cold) pulp test on this tooth. The periapical radiograph also revealed an extended type II invagination from the crown reaching the middle part of root, with no evidence of communication toward the main image of the canal. It also demonstrated an extended radiolucent image at the mesial aspect of the apex (Figure 2). Further radiographic assessment via CBCT was proposed, and subsequently declined by both the patient and her parents. The contralateral tooth (upper left lateral incisor) was extracted one year ago due to the same pathology of development but unsuccessfully treated by the referring doctor. The treatment plan was explained, and written informed consent was obtained to include documentation such as radiographs and clinical photographs for scientific publication. The proposed treatment plan was comprised of two parts: the first being the non-surgical cleaning, shaping and obturation of the conventionally accessible canal spaces, followed by a surgical procedure to access and treat the remaining canal space by root end preparation and filling.\n\nClinical procedure part 1: The affected area was anesthetized by periapical infiltration of 1.8ml of 2% scandicaine Septodont® (Saint-Maur-des-fossés, France) and 1/100.000 vasoconstrictor, followed by isolation of the concerned tooth using rubber dam. The access cavity was achieved aided by a surgical microscope pico ZEISS OPMI® (Jena, Germany), and the high-speed Endo Access Kit® Dentsply-Sirona (Ballaigues, Switzerland), first using a diamond round bur to initiate pulpal exposure, followed by a tapered diamond bur (Figure 3) A Micro-Opener® Dentsply-Sirona (Ballaigues, Switzerland) was used to locate the canal orifice, but dystrophic deposits prevented deeper penetration apically. A # 10 K file Dentsply-Sirona (Ballaigues, Switzerland) was inserted to scout the canal, and working length (WL) was established based on radiographic estimation and clinical tactile feedback. This was in lieu of an electronic measurement; the apex locator failed to demonstrate any patency or continuity indications. Shaping of this part of the canal was completed with a set of S1, S2, F1 and F2 ProTaper® files, Universal Dentsply-Sirona (Ballaigues, Switzerland), in conjunction with copious irrigation with NaOCL. The canal was medicated with a calcium hydroxide temporary dressing AH Temp™ Dentsply-Sirona (Ballaigues, Switzerland). The access cavity was then temporary sealed with a cotton pellet and Cavit™ G (3M ESPE, St Paul, MN, USA). The patient received a prescription of amoxicillin (500 mg orally every 8 hours) and ibuprofen (200 mg every 6 hours). At the second visit, the patient’s symptoms had subsided partially; the patient was not experiencing pain to percussion or palpation; the swelling had disappeared. Removal of the AH Temp™ was accomplished using a K 25 ultrasonic file Irrisafe™ (Satelec, Halifax, Canada) and 2ml of 17% EDTA. After drying the canal space with adequate paper points, a #30 diameter master gutta-percha point from TotalFill® BC (La-Chaud-de-Fonds, Switzerland) was adjusted and sealed in the canal using TotalFill® BC sealer (La-Chaud-de-Fonds, Switzerland) in a single cone technique (Figure 4a).\n\nA tapered round-end high-speed bur placed in the access cavity and an X-ray exposed to make sure of the direction toward the pulp space.\n\n(a) post-operative X-ray after conventional endodontics (b) post-operative X-ray after surgical procedure.\n\nThe surgical procedure of the treatment was scheduled after 2 days. Administration of anesthesia (two cartridges of 1.8 ml of 2% lidocaine with 1: 50 000 epinephrine Dentsply Pharmaceutical, York, PA, USA) was accomplished and a muco-periosteal flap was raised extending from the mesial aspect of tooth #4 to the distal aspect of tooth #8. Under microscopic magnification the apical lesion was observed to be fenestrating the buccal cortical bone. After curettage of the granulation tissue, the refinement of osteotomy, and 1 mm thickness of the root apex resection was executed. Assessment at high magnification (X12) of the resected root surface was conducted to evaluate the extent and completeness of the resection. The root end preparation was created using a diamond coated 4 mm KiS-1D4 (Obtura Spartan, Fenton MO, USA). The prepared apical cavity was filled with Fast set putty TotalFill® BC (La-Chaud-de-Fonds, Switzerland) using the MAP® system (Vevey, Switzerland) for delivery of the apical filling material (Figure 4b). After repositioning of the flap suturing was conducted with 5-0 nylon sutures. The granulomatous soft tissue was harvested and sent for histopathological examination; the biopsy examination confirmed a pathological diagnosis of a periapical cyst. The patient presented 1 week later for control and suture removal with no postoperative pain and uneventful healing. Clinical and radiographic exam of the patient at 6-month intervals exhibited progressive healing of the lesion at recall appointments (Figure 5, Figure 6 and Figure 7).\n\n\nDiscussion\n\nMicrodontia is a condition of one single tooth or more with a size smaller than normal. A peg-shape anomaly is characteristic of a tooth presenting with a crown width at incisal mesio-distal edge smaller than the cervical edge; this abnormal development usually affects upper lateral incisors. This situation may be the source of esthetic and orthodontic concern, and cause a problematic issue for dentists. Peg-lateral prevalence varies between 0.6% and 9.9% depending on ethnicity and sex, however, the overall prevalence reaches 1.8% which is equivalent to 1 in 55 people worldwide12. According to Kim et al. a slightly higher prevalence of peg-laterals and dental anomalies affects females (51.5%)6. In the same study, the authors also concluded that peg-laterals exhibit shorter root lengths compared to normal lateral incisors; they found also an incidence of 19.7% for dens invaginatus. Patients with peg-laterals often complain of esthetic issues, which need treatment orthodontic alignment and/or prosthetic restoration. Coexistence of both anomalies (peg-lateral and DI) would complicate this situation; an effective treatment plan would require endodontic therapy combined with the restorative treatment for esthetic concerns. The scarcity of case reports about microdontia combined with dens invaginatus is remarkable, with only two case reports of this anatomical feature described in the endodontic literature7. Jaikailash et al. reported a five canal peg-lateral treated in nonsurgical approach, with all canals patent from pulp chamber to foramen4. To our knowledge, one single case report has been published using a combined nonsurgical and surgical approach to treat DI affecting a mandibular incisor macrodontia13. If the success of endodontic treatment is dependent upon thorough cleaning of all irritants from canal space, then the complex configuration in DI leaves areas such as fins, communications and cul-de-sac unattainable by conventional instrumentation and disinfecting agents. The presence of a large apical lesion might compromise the conventional drying and sealing of the canal space, requiring a surgical approach to remove the apical lesion and staunch the flow of exudates. In their case report on DI type III with large apical lesion, Falcao et al. stated that, although the complexity of DI configuration could be controlled non-surgically, the authors had to eliminate the apical lesion surgically14. Fregnani et al. also stated that a surgical approach is only necessary in cases where conventional root canal therapy has failed, and in teeth with complex anatomic variations, impeding mechanical access and cleaning of all parts of the canal system. This occurs in some cases of dens invaginatus type III presenting with a periapical lesion15.\n\nThe case here described, had a large area of the canal that was inaccessible by nonsurgical means, thus the combination of non-surgical and surgical endodontics was indicated. The combination of both procedures has been reported to effectively treat cases DI13 with a long term success of 18 years follow-up11. The nature of the root end filling material has been evolving through the last two decades, with ProRooT MTA largely recommended for root end filling13. Recently, FKG (La-Chaud-de-Fonds, Switzerland) has developed a new bioceramic sealer with putty consistency and a fast setting time16. This material is ready-to-use, which eliminates the mishaps of incorrect ratios of powder-to-liquid: mixing errors, and delayed setting. Moreover, the consistency of this material enables easy transport and condensing in the root end preparation, enhancing the quality of the seal and optimizing the tooth prognosis.\n\nBecause of the unusual anatomy of DI, it is recommended to develop a thorough diagnosis and treatment plan, which contributes to the completion of the assessment of the configuration of the canal system16. CBCT exam is particularly helpful for diagnostic purposes and management of teeth presenting a particular anatomy10. However, in the present case report, the radiographic exam was limited to periapical radiographs, which prevented a more complete documentation and visualization of the canal anatomy. Patient and parents signed a written consent to justify their insistence not to proceed with a CBCT examination. Pulp vascularization is also a treatment modality of DI that could not be recommended for the present case17.\n\nThe challenges in treating this case, with a complex anatomy, and an unusual blockage of the canal system, resides in the decision-making; and the ability to perform a conventional approach for treating the accessible part of the canal, and surgically treat the residual portion of the canal. Additionally, the patient feared losing another tooth, providing a further challenge. The contralateral tooth had been extracted 2 years prior, most likely due to the tooth having a similar anatomy resulting in unsuccessful treatment.\n\nTo our knowledge, this is the first case report discussing the use of Fast set putty TotalFill® bioceramics as a retrograde filling material in the treatment of dens invaginatus with 2 years of follow-up. This material has two major advantages compared to MTA; first, the ease of manipulation as a ready mix paste to introduce into the retrograde cavity; second, the prevention of discoloration with the substitution of heavy metallic oxides and ferric oxide which cause discoloration of remaining tooth structure, and thus leads to esthetic failure9.\n\nHowever, this study has a limitation in the radiographic analysis. It would have been more accurate to have a CBCT image as a diagnosis tool in order to elaborate a more adequate treatment plan, knowing the important role of CBCT as a potential aid in management of such cases, the present case has been unfortunately evaluated and negotiated with the aid of conventional radiographs, at the insistence of the patient’s parents.\n\n\nConclusion\n\nAll efforts should be aimed to save permanent teeth in adolescents. Knowledge of the treatment challenges caused by DI, and the use of operating microscope, which has proved to be an invaluable clinical asset, are of great help to control intricacies of complex cases, in both conventional and surgical procedures. The use of newly developed materials with improved qualities and ease of manipulation can enhance the outcomes in challenging cases, such as this case of dens invaginatus and microdontia. The importance of communication with the patient and the parents is of capital importance; it consolidates the relationship and encourages recall visits and long term follow-up.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient and the patient’s parent.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nHülsmann M: Dens invaginatus: aetiology, classification, prevalence, diagnosis, and treatment considerations. Int Endod J. 1997; 30(2): 79–90. PubMed Abstract\n\nAlani A, Bishop K: Dens invaginatus. Part 1: classification, prevalence and aetiology. Int Endod J. 2008; 41(12): 1123–36. PubMed Abstract \| Publisher Full Text\n\nCapar ID, Ertas H, Arslan H, et al.: A retrospective comparative study of cone-beam computed tomography versus rendered panoramic images in identifying the presence, types, and characteristics of dens invaginatus in a Turkish population. J Endod. 2015; 41(4): 473–8. PubMed Abstract \| Publisher Full Text\n\nJaikailash S, Kavitha M, Ranjani MS, et al.: Five root canals in peg lateral incisor with dens invaginatus: A case report with new nomenclature for the five canals. J Conserv Dent. 2014; 17(4): 379–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhan SA, Khan SY, Bains VK, et al.: Dens invaginatus: review, relevance, and report of 3 cases. J Dent Child (Chic). 2012; 79(3): 143–53. PubMed Abstract\n\nKunert GG, Kunert IR, de Figueiredo JA, et al.: Nonconventional Therapeutic Protocol for Type III Dens Invaginatus. J Contemp Dent Pract. 2017; 18(3): 257–60. PubMed Abstract \| Publisher Full Text\n\nZhu J, Wang X, Fang Y, et al.: An update on the diagnosis and treatment of dens invaginatus. Aust Dent J. 2017; 62(3): 261–275. PubMed Abstract \| Publisher Full Text\n\nKato H: Non-surgical endodontic treatment for dens invaginatus type III using cone beam computed tomography and dental operating microscope: a case report. Bull Tokyo Dent Coll. 2013; 54(2): 103–8. PubMed Abstract \| Publisher Full Text\n\nKohli M, Karabucak B: Bioceramic Usage in Endodontics. AAE. Communiqué. 2019. Reference Source\n\nTeixidó M, Abella F, Duran-Sindreu F, et al.: The use of cone-beam computed tomography in the preservation of pulp vitality in a maxillary canine with type 3 dens invaginatus and an associated periradicular lesion. J Endod. 2014; 40(9): 1501–4. PubMed Abstract \| Publisher Full Text\n\nWayama MT, Valentim D, Gomes-Filho JE, et al.: 18-year follow-up of dens invaginatus: retrograde endodontic treatment. J Endod. 2014; 40(10): 1688–90. PubMed Abstract \| Publisher Full Text\n\nKim JH, Choi NK, Kim SM: A Retrospective Study of Association between Peg-shaped Maxillary Lateral Incisors and Dental Anomalies. J Clin Pediatr Dent. 2017; 41(2): 150–3. PubMed Abstract \| Publisher Full Text\n\nZhang P, Wei X: Combined Therapy for a Rare Case of Type III Dens Invaginatus in a Mandibular Central Incisor with a Periapical Lesion: A Case Report. J Endod. 2017; 43(8): 1378–1382. PubMed Abstract \| Publisher Full Text\n\nFalcao Lde S, de Freitas PS, Marreiro Rde O, et al.: Management of dens invaginatus type III with large periradicular lesion. J Contemp Dent Pract. 2012; 13(1): 119–24. PubMed Abstract \| Publisher Full Text\n\nFregnani ER, Spinola LF, Sônego JR, et al.: Complex endodontic treatment of an immature type III dens invaginatus. A case report. Int Endodont J. 2008; 41(10): 913–919. PubMed Abstract \| Publisher Full Text\n\nJitaru S, Hodisan I, Timis L, et al.: The use of bioceramics in endodontics - literature review. Clujul Med. 2016; 89(4): 470–473. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang J, Zhao Y, Qin M, et al.: Pulp revascularization of immature dens invaginatus with periapical periodontitis. J Endod. 2013; 39(2): 288–92. PubMed Abstract \| Publisher Full Text" }	[ { "id": "74370", "date": "04 May 2021", "name": "Tariq Yehia", "expertise": [ "Reviewer Expertise Endodontics & Micro-endodontics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTitle: well-expressed.\nIntroduction: Good, relevant, well-referenced.\nCase report: The following should be explained by the author:\nThe mechanical prep. of the accessable coronal segment of root canal system reached only # F2 Protaper as MAF, does the author think that this was an appropriate size regarding that this was only in the large coronal part? Please discuss this in the discussion section.\n\nWhat was the rationale behind using single cone with bioceramic sealer in this large coronal part of the canal? Why did you not consider injection technique especially that you will move to apical surgery afterward. Please comment on this in the discussion section.\n\nWhat was the type of mucoperiosteal flap used. Please add to text.\n\nIt will be preferable if composite intraoperative photographs figure can be added.\n\nThe authors mentioned that the length of the ultrasonic tip used was 4mmm however, the length of the retrofilling appears to be longer. Can you discuss this in the discussion section?\nDiscussion: well-presented & properly referenced.\nReferences: relevant & updated.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "84332", "date": "14 May 2021", "name": "Sidhartha Sharma", "expertise": [ "Reviewer Expertise Endodontics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe main query lies in the methodology. The author mentions that the large area of the canal was inaccessible by nonsurgical means, thus the combination of non-surgical and surgical endodontics was indicated. The preoperative x ray clearly shows a wide root canal and a type II invagination and still the main canal could not be negotiated under magnification. The operator may have found the invagination and hence could not negotiate it since it was a type II. Was the main canal missed? Please justify. An intraoral pic of the canal orifices after access opening is required. Had the main canal been cleaned and shaped, it is possible that a surgical treatment would not have been required unless there is an apicomarginal defect. Combination of treatment is often required in type III DI where the invagination communicates with the periapical area. Clinical picture of the surgical area after flap elevation is required. Was there any apicomarginal defect? Any bonegrafts /GTR applied? The length of retrofill is more than the 4 mm retropreparation? Kindly explain.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-125
https://f1000research.com/articles/8-1830/v1	30 Oct 19	{ "type": "Opinion Article", "title": "Advocate cultivation of academic ethics: why is it necessary?", "authors": [ "Sok-Ja Janket", "Jukka Meurman", "Eleftherios P. Diamandis", "Sok-Ja Janket", "Jukka Meurman" ], "abstract": "We teach and practice ethical behavior with all clinical and research activities. Notably, we are well educated to treat the subjects participating in research studies with high ethical standards. However, the ethics of interacting with colleagues, or with junior faculty members, are neither well defined nor taught. Dealing with junior faculty has parallels to dealing with vulnerable research subjects such as children, mentally or physically challenged groups, prison inmates or army recruits. Like any other vulnerable population, lower-ranking faculty members are often at the mercy of department chairs or other higher-ranked faculty members. Herein we present some potentially unethical or unfair examples related to academic research. Our goal is to educate the academic community of conceptual paths and to prevent similar untoward occurrences from happening in the future. Unethical behaviors related to sexual misconduct have already been described elsewhere and are not included in this manuscript.", "keywords": [ "Academic", "Ethics", "Behaviour", "Education", "Faculty" ], "content": "Introduction\n\nIn 2014, the World Medical Association celebrated the 50th anniversary of the Declaration of Helsinki1,2. This Declaration forms the basis for all later ethical proclamation relating to how “we care for the persons who are our patients, for those who seek advice, and protect those who volunteer in research studies”2. Through repeated training in human research subject protection, researchers recognize the malevolence of exposing unsuspecting prison inmates to syphilis bacteria to study immune responses or use organs from executed criminals for transplantation without their consent. However, the boundaries of ethics in collaboration with colleagues or with the junior faculty members are not well-defined nor discussed. We, hence, discuss the importance of teaching and following a code of ethics in interactions between colleagues in the academic setting.\n\nOne of the pioneers of ethical education, Dr. Bertolami, wisely pointed out that our ethics curricula in biomedical education are informative, but fail to be formative and transformative3. The reason is that ethics courses teach mainly abstruse concepts that are vague and intangible. Thus, as he asserts, they stay informative at best but are not strong enough to change a person’s views. Indeed, “ethics” is often used interchangeably with “morality”4, but ethics has more to do with a “philosophical concept” and morality has more to do with human actions5. Aristotle used the word “hexis” to describe moral virtue in relation to ethics6. Because hexis is an active condition, “virtue”, in this context, implies an action. Again, we arrive at the conclusion that ethics must bring action, namely being transformative7. Therefore, it is necessary to teach ethics in a more action-based and example-based format so that learners can apply ethical judgment in their everyday activities.\n\nDr. Bertolami also presented the Aristotelian grouping of an ethical bell curve7. We created a similar bell curve using our data and labeled them according to Aristotelian/Bertolamian categorization (Figure 1). We surmise that very few individuals in health science belong to the ‘vicious’ group, where serial killers or white-collar embezzlers may be included, or the ‘paragon’ group where Mother Theresa-like persons will belong. We arbitrarily consider them outliers belonging to the upper and lower 5% and also speculate that most researchers belong in the middle 90%7. Thus, the ethical transgressions we observe in biomedical research fall into the broad area of the middle 90% and it is not easy to determine whether the observed conduct is truly unethical, marginally unethical or not unethical. Clearly, there is a need to define the boundaries between ethical and unethical conduct in academia and give actual examples that underscore such demarcation. We suggest that special committees be formed for each academic institute whose responsibilities are to oversee any deviations from the acceptable ethical boundaries and make recommendations to remediate any alleged ethical transgressions. Let us explore some examples derived from our own experiences and consider solutions to resolve or prevent similar incidences. We have summarized our experience in academic ethics and suggested remedies to prevent future unethical transgressions in Table 1.\n\nThis graph was modified with permission from Bertolami7.\n\n\nEpisode 1a: “Challenging your senior faculty could be hazardous to your career!”\n\nDr. Sackett humorously described8 how senior faculty members can wield undue power over lower-ranking members of academia beyond the scientific context8. This is more palpable when the advisees reveal the flaws of senior members’ research methodology8. One example of such concealed retaliation can be found in the case of Researcher A.\n\nIn late 1996, several small dental studies published a significant association between periodontitis and cardiovascular diseases (CVD)9,10. Admittedly, these are clinical studies fraught with varying degrees of deficiencies and small sample sizes. Interestingly, two large studies from a famous institution (θ University) were published with null results (no association). Thereafter, the whole world was eager to believe the null results because of the institution’s fame, seemingly excellent methodology and the large sample sizes (~55,000 and ~22,800 study subjects).\n\nWhile learning meta-analysis methodology as a Master of Public Health student at the same famous θ University, Researcher A chose this topic as a class project to evaluate the evidence without praejudicium (prejudged or pre-decided without being fairly heard). Researcher A truly believed that making a clear objective conclusion would benefit the research community and his faculty advisors would be pleased with such an endeavor. Researcher A did the project so well and received a standing ovation from fellow classmates and high praise from the instructor. The results were “pure science” without any influence of personal feelings or sense of affiliation. The conclusion was that there was a small but significant association between periodontitis and CVD. More importantly, the mathematical analyses have proven that seemingly poorly conducted studies overestimated the risk by only 13%, while ostensibly well-conducted studies underestimated the risk by 29.7%, thus appearing to be more biased than smaller studies. Later, Researcher A informed his faculty advisor of the results of meta-analysis. This faculty adviser (FA 1) was one of the lead authors of the null-result reporting papers from the θ University. FA 1 showed Researcher A the doctoral thesis which reported similar 30% attenuation in the risk estimates some 4 years earlier. Thus, what Researcher A observed in the meta-analysis was in agreement with the faculty adviser’s thesis. We note that the said faculty adviser did not realize the deficiencies of own thesis which reported 30% underestimation of the true risk. Rather, this faculty adviser thought that the culpability resided in other studies. Consequently, this faculty member wrote several opinion papers criticizing the other small studies.\n\nSince the publication of this meta-analysis, which became a sentinel study cited over 560 times, Researcher A’s manuscripts and grant proposals have been harshly criticized and rejected numerous times presumably by these faculty advisors. Because of their institutional fame, these faculty advisors benefited from the privilege of being the most sought-after reviewers. After being rejected so many times, Researcher A’s grant was enigmatically funded when these faculty advisors were facing a legal action from an anti-compound λ group.\n\n\nEpisode 1b: How to reconcile when student’s research collides with the advisers’ theory?\n\nResearcher E was a classmate of Researcher A at the same θ university and both were mentored by the same faculty advisors. Researcher E collected data for over 5 years and analyzed them. Researcher E found that her results suggested that exposure to compound λ increased the risk of osteosarcoma in adolescent boys. When Researcher E was about to publish the results, said advisors strongly recommended not publishing them citing that her data were of poor quality. This obstruction of the doctoral thesis by said faculty advisors leaked to an advocacy group opposed to the use compound λ and public uproar followed. Moreover, θ University, fearful of any future litigation from the anti-compound-λ group, forced the senior of the two faculty advisors (FA 2) to retire. Collaterally, the poor data collection method as revealed by meta-analysis prevented the younger of the two faculty advisers from winning tenure. Although neither Researcher A nor Researcher E had anything to do with the anti-compound-λ group’s protest on these faculty advisers, the animosity from the faculty advisers towards Researchers A and E deepened without due cause. Although the results showing the association of compound λ and the risk of osteosarcoma were finally published, Researcher E could not find a research position after 6 years of intensive PhD training. Perhaps, the behind-the-scenes evaluation might have had something to do with this researcher’s thwarted research career.\n\nHere, it is worth discussing the objectivity of covert consultation of mentors without the knowledge of mentee’s especially when mentees have differing scientific views from that of their mentors. It is common practice that prospective employers, including post-doctoral research positions, contact the former employer or former faculty advisors about the prospective employee. The sad part is that this important process takes place without the knowledge of the researchers seeking positions. Thus, the nascent researchers have no idea why their job applications were declined. Consequently, what Voltaire had said some 300 years ago was proven true in 2000s: “It is dangerous to be right in matters on which the established authorities are wrong.”\n\nOur perspectives:\n\nTo strengthen the fidelity of science, differing opinions should be encouraged. This includes divulging the flaws of faculty members’ methodologies.\n\nWe find the action of a prospective employer contacting faculty advisor without informing the position seeking researchers unethical although it is quite prevalent. If such an action is elected, they should contact the mentor in the presence of the mentee. Researcher A did not know why his numerous job applications were rejected until one prospective employer called the FA1 in the presence of Researcher A. We find this covert evaluation beyond the written letter of recommendation is despicable and should be abolished.\n\nWe recognize that it is only human to harbor ill-feelings towards the mentees who exposed one’s own research flaws. We, therefore, suggest that mentors should totally extricate themselves from giving any evaluations of those mentees who provoke ill-feelings. In the case of these two faculty advisors, the younger FA1 delegated reviews of the mentees to the senior FA2 who is more prone to be harsher due to their powerful positions. Technically, FA1 relinquished review of mentees but handed it to a crueler evaluator.\n\nAlso, take note that until the publication of the said meta-analysis, no one understood the cause for the conflicting results in the periodontitis-CVD association although all the researchers involved were top-notch scientists. This becomes another topic of unethical behavior by another researcher in Episode 2.\n\n\nEpisode 2: Do two bad ethical transgressions cancel each other out?\n\nAs stated above, no one who read the studies reporting the association between periodontitis and CVD knew the cause for the conflicting reports. When Researcher A had proved that non-differential misclassification was the cause of the null results using meta-regression, many people had an “aha!” moment. An “aha!” moment occurs when a simple principle that many people already know explains a complex phenomenon. For this reason, it is easy to claim another person’s intellectual property (IP) that brought an “aha!” moment as their own.\n\nIn essence, Researcher A proved that the two large studies with meticulous confounding adjustment were more biased than the small, seemingly poorly conducted studies. Because it is expensive to conduct clinical examinations when a sample size is in the tens of thousands, the larger studies used a questionnaire to diagnose periodontitis. When a less-than-precise method, such as a questionnaire, is used, it causes non-differential misclassification. Non-differential misclassification is an elementary epidemiologic concept that can be explained in simple terms as follows: The truly diseased are included in the non-diseased group and some non-diseased persons are included in the diseased group because the measuring instrument was not precise. The result is reduced contrast which, in epidemiology, is referred to as “attenuation of the risk estimate”. It is a well-known epidemiologic principle that non-differential misclassification moves the risk estimates towards the null. However, connecting the dots of non-differential misclassification to the results of the “periodontitis-CVD relationship” was Researcher A’s IP. Let us peruse what has transpired after Researcher A’s meta-analysis.\n\nResearcher A wrote in the meta-analysis in 2003: “…potential for underestimation by 2 large epidemiologic studies because of the non-differential misclassification stemming from the use of questionnaires instead of clinical examination of periodontal disease.”\n\nAuthor D wrote quoting Researcher A (not for non-differential misclassification but for heterogeneity) in 2005: “potential misclassification of periodontitis in studies not employing periodontal probing for assessment of periodontitis……. Indeed, the attenuation of relative risk estimates due to such misclassification can be quite dramatic”.\n\nIn 2008, Author D wrote quoting his own 2005 publication: “potential misclassification of periodontitis in studies not employing periodontal probing for assessment of periodontitis…Indeed, the attenuation of relative risk estimates due to such misclassification can be quite dramatic”.\n\nCareful readers will recognize that Researcher A in 2003, and author D in 2005, wrote the same scientific construct with small changes in wording. Is Author D not obligated to quote Researcher A for this sentence because Researcher A has already published the very concept 2 years prior? Clearly, Author D had read Author C’s article when he quoted Researcher A for “heterogeneity” which is a far less important piece of information than misclassification. It appears that Author D used a two-step “bait and switch” tactic to divert the contest from Researcher A by quoting for something peripheral to the issue and made the more important concept of “non-differential misclassification” in the periodontitis-CVD relationship as his own IP.\n\nResearcher A read the above phrase which Author D wrote in 2008 and thought “Whoa, this is exactly what I wrote in 2003. Did he quote me”? But, it was found that Author D quoted his own 2005 paper. So Researcher A traced back to his 2005 publication and discovered that Researcher B quoted Researcher A for very nonessential “heterogeneity” but not for the important “non-differential misclassification”. A majority of meta-analyses report heterogeneity. Thus, it appeared that the construct of “non-differential misclassification attenuated the risk estimate in the periodontitis-CVD association” became his own IP in a two-step ‘bait and switch’ tactic. First, neutralize any protest from Researcher A by quoting the article for an unimportant fact and leaving the real important construct out. And in the second step, he quoted his own paper reporting the important construct without citing the Researcher A, who published this concept some years earlier. This two- step unethical misappropriation of someone else’s IP could be due to unintentional neglects. This is why we need ethical training to recognize that a subtle transgression may be a form of plagiarism.\n\nOur perspectives:\n\nWe must educate ourselves to give credit to prior studies. Even if the concept involves a basic and mundane principle, the first person who used this mundane construct to solve the scientific queries should be given credit.\n\nWe need to recognize that ethics is a human construct that needs to be taught.\n\nEthical learning curve is applicable to everyone. We first thought citing Aristotle’s Nicomachean Ethics would be adequate citation for our categorization of human ethics (Figure 1). However, after further thinking, we determined citing Bertolami would be appropriate because he used the modern terminologies which made us easy to understand Aristotle’s categorization.\n\nWe advocate that plagiarism should include not only “word for word” copying but also using other people’s concepts and ideas without appropriate citation.\n\nLet us consider another example of a study using a mundane fact to explain a weighty conclusion. In 2012, a study conducted by a group of physicians and epidemiologists reported in a leading medical journal that dental x-rays might increase brain cancer meningioma in children11. Later, a group of investigators reported that the exposure assessment of the original study11 was done by self-report and thus the results are not trustworthy12. Almost everyone with elementary knowledge in epidemiology knows that self-reported data are notoriously unreliable. Do we see the parallels of flawed methodology in the usage of questionnaire and self-report? They are both extremely imprecise, causing biased results. Anyone who has basic epidemiologic knowledge and read the original article11 would have recognized the flawed exposure assessment. Nevertheless, it is our opinion that the first researcher(s) who identified and published this flaw in the original study11 should be given credit as the owner of that IP12.\n\n\nEpisode 3: Watch your foes carefully and watch your friends even more carefully!\n\nResearcher C has been regarded as an expert on the relationship between oral infections and CVD and had published over 30 papers on the topic. Researcher C’s mentor, Dr. Z, known for her fairness and thoughtfulness helped Researcher C’s career transition from clinician to researcher. In appreciation of this mentor’s guidance, Researcher C included the mentor in a number of oral health and CVD-related publications. Subsequently, Dr. Z decided to write a grant proposal for assessing the relationship between oral health and CVD, assuming the role of the principal investigator (PI). In essence, the mentor became Researcher C’s competitor. The mentor appears to think that including researcher C as a co-investigator is fair compensation. However, Researcher C felt that Researcher C should be one of the co-PIs. The differing views on fair compensation caused a rift in their once close relationship.\n\nSadly, ideas, and the associated IP can easily be purloined without any credit given to the source during the discussion because copyright or patent is not possible for these ideas. The concepts Researcher C shared with the mentor during a conversation became the mentor’s idea without any credit given to Researcher C. The concept that forms IP is the fundamental starting point for any scientific endeavor and should come from the PI, not the co-investigators. Often, in open discussions, Dr. Z requested members to provide opinions on how to approach the methodology. This is particularly troublesome when senior faculty members urge junior faculty members to donate their IP although the senior faculty members are the principal investigators. If junior faculty members decline, the senior faculty members are in the position of negatively affecting the junior faculty members’ careers. In this context, the junior faculty members are no different than the vulnerable research study subjects. Therefore, every effort should be made to protect junior faculty members in research collaborations.\n\nOur perspectives:\n\nAn unspoken rule in research is that mentors should stay clear of the mentee’s research domain. This is because mentors are in a position of power and if they compete against mentees, they are in more advantageous locus. A good mentor will place mentees as PIs and put themselves as co-investigators.\n\nIf the senior faculty is the PI, their role is to develop a study rationale and the research strategy. This requires much reading and critically evaluating prior studies not only supporting his/her study objectives but opposing said objective. The role of PI (i.e., building a study rationale and planning the execution strategy of the rationale) should never be delegated.\n\n\nEpisode 4: Ethics in grant proposal writing\n\nCurrently, few researchers consider ethics when writing grant proposals. However, this should change, because a poorly conceived study will waste limited research resources and reduces the probabilities of other worthy studies getting funded. Thus, all researchers have a collective responsibility to conceptualize, prepare and execute a study plan that will benefit society.\n\nMost researchers are under tremendous pressure from the institution with which they belong and “getting funded” becomes the ultimate impetus for writing grants without adequate learning and preparation. What was once, “publish or perish”, nowadays becomes “funded or fade away”. In these conditions, the foremost role of the principal investigators (PIs) is accumulating adequate knowledge and developing a rationale so that the co-investigators will follow the well-planned study protocol. Too often, PIs are senior faculty members and sometimes have minimal knowledge of the study’s objectives. Using the position of authority, they delegate important tasks that clearly belong to the role of PI to junior faculty members who do not have enough experience or knowledge. The consequence of this dereliction of duty is an ill-prepared grant that might waste resources.\n\nThe vertical relationship between senior and junior faculty members in collaboration harbors potential for an abuse of authority. The delegation of tasks can be perceived as an order. Often, senior researchers use words to appeal to the guilty conscience of junior faculty members, such as “this work is good for all of us and good for the institution”. Basically, this can be interpreted as “work hard without getting the credit you deserve”. The unspoken message is “if you do not do what I ask you to do, you are acting against all of us and against the organization we serve.” Forced donations, as suggested by senior faculty members, whether monetary or IP, should be discouraged. We decry sweat shops in South East Asia for their abusive labor practice but very few are speaking against intellectual sweat shops in academia. Is it because the perpetrators and victims both belong to the highly educated echelons of society? Abusive labor practice is abusive, no matter where it occurs.\n\n\nEpisode 5: Ethical issues in grant review\n\nThere are additional problems with the grant review process. The reviewers are usually those who were funded in the similar domain of research and they often have a hidden agenda to fund the application in the same direction as their own. For example, if the reviewer received funding to investigate oral cancer in relation to human papilloma virus (HPV), it is highly unlikely that this reviewer will fund an application aiming to prove that HPV was not a cause of oral cancer. Thus, whatever biases the reviewer has, will be perpetuated and any proposal that is not concurrent with reviewer’s research direction will likely not be funded. These biases are often unconscious, and the reviewer may not realize that she/he may be blocking a new thesis. Although the National Institute of Health (NIH) is looking for “innovative” research, our experience tells us that reviewers rarely support any innovative idea that may take away their own funding potentials. Others have expressed similar concerns on these non-financial conflicts of interest13. This is a clear conflict of interest but the grant review system has been in operation as far as we can remember. Thus, we do not know whether the application has no scientific merit or is opposite of the reviewer’s research theory.\n\nThe qualifications of reviewers can also sometimes be problematic. If a study involves human-level outcomes, it will be reviewed by epidemiologists who usually do not conduct molecular-level research. And yet, their review determines the fate of many well-thought-out translational research projects. Often, the value of the scientific merit is secondary to the submitter’s personality. Someone who never spoke against another scientist’s flaws will get funded with mediocre science, while scientists who tried to expand our scientific understanding by pointing out deficiencies in other’s research may not be so lucky.\n\nWasteful funding may be attributed to the funding agencies’ demand for “innovation” as a key element in grant proposals. “Innovative” research innately encourages charting untested waters. At present, buzzwords ending with “-ome”, i.e., genome, microbiome, proteome, transcriptome, metabolome, etc. are in vogue. These studies are looking at the whole collective constructs, be it microorganisms (over 10 trillion in a human body), genes (25,000+ human genes) or proteins (10,000 to potentially billions in a human body). First, it is nearly impossible to pinpoint any one or several of these as causative of a human pathology. Moreover, the investigators use an easy to access compartment such as the fecal microbiome to estimate the causative microbes in the gut, which may not be similar14. This is a major scientific misstep because feces are the end results of intestinal activities, not the cause for them15,16. Consequently, many millions of research dollars have been spent but we are not certain of the benefits these results may bring to society. Reviewers will not support a brand new idea citing “the lack of preliminary data” even in pilot grants. Without a pilot funding, the idea will not be tested at all. The ideas with proven track record will be shunned as “not innovative enough.” Once again, Voltaire was correct in saying: “When the idea is new, people say it is not true. When finally it is proven to be true, people say it is not new.”\n\nOur perspectives:\n\nThe key ethical consideration in grant writing should be whether the grant proposal will improve human health. Thus, selecting causal relationship that will engender better health for all humans is the crucial.\n\nThe most important tenets of ethical grant review is reviewing the potential for human health improvement. Too often, many studies that will never reach human level results guzzle up a major portion of research funds.\n\nThere is no easy way to protect the rights of junior faculty members when they collaborate with senior investigators. Educating scientific community on this issue and the senior members of faculty may voluntarily protect the junior faculty members.\n\n\nConcluding remarks\n\nWe must all take pride in not surrendering to the overwhelming power of the established dogma. As the late columnist Charles Krauthammer said “If you are going to leave the medical profession because you have something to say, you betray your whole life if you don’t [say] and if you don’t say it honestly and bluntly.” For all those who spoke out to make academic research just and fair, whether their honesty and bluntness will contribute to the betterment of academic research ethics will not be known immediately. Only history will tell.\n\n\nData availability\n\nNo data are associated with this study.", "appendix": "References\n\nWorld-Medical-Association: World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. Jama. 2013; 310(20): 2191–2194. PubMed Abstract \| Publisher Full Text\n\nParsa-Parsi RW, Ellis R, Wiesing U: Fifty years at the forefront of ethical guidance: the world medical association declaration of Helsinki. South Med J. 2014; 107(7): 405–406. PubMed Abstract \| Publisher Full Text\n\nBertolami CN: Why our ethics curricula don't work. J Dent Educ. 2004; 68(4): 414–425. PubMed Abstract\n\nDeigh J: Empathy and universalizability. Ethics. 1995; 105(4): 743–763. Reference Source\n\nWahlman-Calderara T, Meurman JH: Ethics in Biomedical Research. In: Translational Oral Health Research. (ed. Meurman, J.H.) (Springer International Publishing AG, 2018). 159–168. Publisher Full Text\n\nSachs J: Internet Encyclopedia of Philosophy. In: Aristotle/Ethics. 2019. Reference Source\n\nBertolami CN: Moving Ethics Curricula Forward. Ethics in Biology, Engineering & Medicine - An International Journal. 2011; 2: 87–106. Publisher Full Text\n\nSackett DL: The sins of expertness and a proposal for redemption. BMJ. 2000; 320(7244): 1283. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMattila KJ, Nieminen MS, Valtonen VV, et al.: Association between dental health and acute myocardial infarction. BMJ. 1989; 298(6676): 779–781. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeck J, Garcia R, Heiss G, et al.: Periodontal disease and cardiovascular disease. J Periodontol. 1996; 67(10S): 1123–1137. Publisher Full Text\n\nClaus EB, Calvocoressi L, Bondy ML, et al.: Dental x-rays and risk of meningioma. Cancer. 2012; 118(18): 4530–4537. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDirksen D, Runte C, Berghoff L, et al.: Dental X-rays and risk of meningioma: anatomy of a case-control study. J Dent Res. 2013; 92(5): 397–398. PubMed Abstract \| Publisher Full Text\n\nAbdoul H, Perrey C, Tubach F, et al.: Non-financial conflicts of interest in academic grant evaluation: a qualitative study of multiple stakeholders in France. PLoS One. 2012; 7(4): e35247. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBajaj JS, Hylemon PB, Ridlon JM, et al.: Colonic mucosal microbiome differs from stool microbiome in cirrhosis and hepatic encephalopathy and is linked to cognition and inflammation. Am J Physiol Gastrointest Liver Physiol. 2012; 303(6): G675–685. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJanket S, Nunn ME, Salih E, et al.: Evidence-based approach in transaltional dental research. In: Translational Oral Health Research. (ed. Meurman, J.H.) (Springer International Publishing 2018). Publisher Full Text\n\nJanket SJ, Ackerson LK, Diamandis EP: Gut Microbiotas and Immune Checkpoint Inhibitor Therapy Response: A Causal or Coincidental Relationship? Clin Chem Lab Med. 2019. PubMed Abstract \| Publisher Full Text" }	[ { "id": "56143", "date": "25 Nov 2019", "name": "Konstantinos Drosatos", "expertise": [ "Reviewer Expertise Metabolic Biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, Sok-Ja Janket et al. present several hypothetical cases that resemble real-life situations and pertain to ethical challenges that researchers deal with on frequent basis primarily at the mentor-mentee level. The authors are experienced researchers with significant training record. Thus, both their experience and academic caliber render them suitable to offer unbiased and objective point of view on such an important matter.\nHere are my suggestions that may improve the content of the article:\nThe use of single letters as hypothetical names is somewhat confusing, especially because multiple exams are listed in the text. Instead of using Researcher Dr. A, it would be easier to process designations such as Dr. Mentor, Dr. Postdoc, Ms. Student etc.\n\nAlthough it is understandable that not all paradigms can be included in the article, it would be important to add one more scenario on \"Ethics in assigning leadership positions to junior faculty\" e.g. The Society of Research is creating an Early Career Committee to address aspects that pertain to junior faculty. The leadership of the Society of Research has to nominate junior members to operate the ECC. Dr. Past Trainee, who had pursued his post-doctoral training with the President of the Society of Research is selected instead of Dr. Award, who received last year's Outstanding Junior Faculty Award... [CONTINUE]\n\nThe article would have been even more meaningful if the authors devoted a section on certain and clearly described suggestions about institutional action that needs to be taken. The section should go beyond description of principles and include even some examples of action that has been taken by leadership to serve ethical standards and the authors may be aware of.\n\nI am not positive of the suggestion that research proposals should be reviewed in the light of potential for improvement of human health. Various research findings, such as siRNA that was discovered in plants1 were translated to health-related applications long time after their discovery, which might not even imply translatability.\n\nMINOR: p.6; Typo in Episode 2: Replace \"had proved\" with \"had proven\"\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "56447", "date": "20 Dec 2019", "name": "Julie Shaw", "expertise": [ "Reviewer Expertise point-of-care testing" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHere, the authors describe several ethical considerations in academics. This is an interesting topic and the ethical issues related to academic are seldom acknowledged. This is largely an opinion piece, with the conclusions those based on the authors' opinions. Unconscious bias in academics is an important topic that should be addressed.\nMy only comment for consideration to address is for the authors to somehow make clearer the relationships they describe in their \"episodes\". I found it difficult to keep track of the various relationships. Perhaps there is a way to do the pictorially.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1830
https://f1000research.com/articles/8-890/v1	20 Jun 19	{ "type": "Systematic Review", "title": "The effects of interventions preventing self-harm and suicide in children and adolescents: an overview of systematic reviews", "authors": [ "Ida Sund Morken", "Astrid Dahlgren", "Ingeborg Lunde", "Siri Toven", "Astrid Dahlgren", "Ingeborg Lunde", "Siri Toven" ], "abstract": "Background: Self-harm and suicide in children and adolescents are of serious consequence and increase during the adolescent years. Consequently, there is need for interventions that prevent such behaviour. The objective of this paper: to evaluate the effects of interventions preventing self-harm and suicide in children and adolescents in an overview of systematic reviews. Methods: We conducted a review of systematic reviews (OoO). We included reviews evaluating any preventive or therapeutic intervention. The quality of the included reviews was assessed independently, and data was extracted by two reviewers. We report the review findings descriptively. The certainty of the evidence was assessed using Grading of Recommendations Assessment, Development and Evaluation (GRADE). Results: Moderate certainty evidence suggests that school-based interventions prevent suicidal ideation and attempts short term, and possibly with long term effects on suicide attempts. The effects of community-based interventions following suicide clusters and local suicide plans are uncertain, as are the benefits and harms of screening young people for suicide risk. The effects of most interventions targeting children and adolescents with known self-harm are uncertain. However, low certainty evidence suggests that dialectical behavioural therapy and developmental group therapy are equally as effective on repetition of self-harm as enhanced treatment as usual. Conclusions: Research on several recommended practices, such as local suicide plans, prevention of suicide clusters and approaches to risk assessment, is lacking. When implemented, these interventions should be closely evaluated. There also is need for more research on treatment for repeated self-harm, including long term follow-up, and in general: possible harmful effects. Policy makers and health providers should consider evidence from population-based studies and adults in preventing self-harm and suicide in children and adolescents. Also, approaches showing promise in treatment of conditions associated with self-harm and/or suicidality, such as depression and psychosis, should be considered. PROSPERO registration: CRD42019117942 08/02/19.", "keywords": [ "Self-harm", "Suicide", "Adolescents", "Children", "Mental health", "Prevention", "Treatment", "Evidence-based practice" ], "content": "Introduction\n\nSelf-harm involves intentional self-poisoning or self-injury, irrespective of type of motive or the extent of suicidal intent1,2. It is often a coping mechanism used to solve a difficult situation and can serve several functions. Affect regulation, managing painful unpleasant emotional states including making emotional pain physical and blocking bad memories, is commonly reported3. Self-harm can also serve interpersonal functions, such as seeking help from someone or communicating the extent of pain3. In addition, people who self-harm sometimes report self-punishment as a motivation3. Completed suicide is defined as the act of intentionally ending one’s own life4. Self-harm and suicide result from underlying factors such as other mental health problems, exposure to traumatic events or other difficult circumstances in the young person’s environment. Exposure to family and/or friends self-harm and suicide may contribute to self-harm and suicide in adolescents, a phenomenon referred to as “social contagion”5.\n\nSelf-harm is prevalent among adolescents6. Due to few studies on self-harm in individuals younger than 12 years, it is hard to estimate the prevalence of self-harm in children in the community. However, presentations to hospital after self-harm are rare in this age-group5. Across international studies, 18% of adolescents between the ages of 12 and 18 report a history of one or several episodes of intentional self-harm. Prevalence is highest amongst adolescent girls, but it is also a problem amongst boys7. Some studies indicate that the gender differences are smaller than previously assumed, and that boys often inflict self-injury in other ways than girls; while girls often cut themselves, boys more often hit themselves8. Self-harm may be a temporary or more long-lasting in nature7, and one episode of self-harm is a strong predictor of repetition of this behaviour9,10. When self-harm is repeated, the person often advances to a combination of different methods, increasing the medical severity11. Suicide is on the other hand rare before the age of 15 but increases in prevalence through adolescence6. In most parts of the world, male adolescents are more likely to commit suicide than female adolescents12. It is the most common cause of death in female adolescents, and the third most common cause of death in male adolescents (after road-traffic accidence and violence)6. As such, that there is clearly a need for effective prevention of self-harm and suicide in children and adolescents.\n\nSeveral reviews of interventions for preventing self-harm and suicide exist. However, many are of variable quality, or are outdated13–18. As is the case for many health conditions, there is a large overlap in topics covered by the reviews, making it difficult for professionals to sort out the best available evidence in making informed decisions19. Consequently, we wanted to provide an up-to-date overview of the best quality summarized evidence of effects of interventions aimed at preventing self-harm and suicide, supporting informed decision-making.\n\nThe objective of this review is to summarize the effects of interventions for preventing self-harm and suicide in children and adolescents.\n\n\nMethods\n\nThis review was registered with the international prospective register of systematic reviews (PROSPERO; CRD42019117942) on 08 February 2019.\n\nWe included systematic reviews published in 2012 and later (last date searched August 2018), with publications in English, Norwegian, Danish or Swedish, and fulfilling the DARE-criteria20. The inclusion criteria (PICO) is presented in Box 1.\n\nWe excluded systematic reviews that did not meet the criteria for the above-mentioned PICO:\n\n• Children and adolescents with other main-diagnosis, e.g. children admitted to hospitals because of somatic illness at the same time as experiencing depressive symptoms.\n\n• Interventions preventing other behaviours with no direct association with mental health, e.g. interventions targeting smoking cessation.\n\n• Pharmaceutical interventions compared to placebo. This review was conducted to inform decision-making in Norway, and for this purpose only direct comparisons between pharmaceutical treatments were judged to be relevant.\n\nThe literature search for this review was completed in August 2018 and is largely based on IN SUM: a database of systematic reviews on effects of child mental health and welfare interventions21. IN SUM indexes reviews related to children’s and young people’s mental health from the following databases: Cochrane Database of Systematic Reviews, Campbell Library, PsycINFO, MEDLINE, Embase, Web of Science, Database of Abstracts of Reviews of Effects (DARE) and Evidence Based Mental Health. (see extended data22 for a description of the IN SUM search strategy).\n\nThe present review of systematic reviews was developed following the principles of the Cochrane handbook23. Two researchers independently reviewed all publications indexed in IN SUM (two of the athors: AD or ISM, and/or a research colleague KTH). We also hand-searched for relevant systematic reviews, in the following databases and organisations:\n\n• The Norwegian Institute of Public Health\n\n• The Swedish agency for health technology assessment and assessment of social services(SBU)\n\n• The Norwegian Directorate of Health\n\n• The Danish Health Authority\n\n• The National Institute for Health and Care Excellence (NICE)\n\nAll publications judged to meet the inclusion criteria were retrieved in full text. Two researchers (ISM, AA) independently screened and assessed all full text publications for potential inclusion. In cases of disagreement, we consulted a third person.\n\nWe sorted all included reviews by population and which interventions were compared (the PICOs). In cases were more than one review addressed the same treatment comparison for the same population, we included the review with the newest search (and completeness of this search by considering the included studies) and the best quality. In considering overlap, the first author (ISM) extracted this information from the reviews and the second author (AA) double-checked this information. Further, we assessed the quality of the included reviews based on a checklist for systematic reviews (AMSTAR: A MeaSurement Tool to Assess systematic Reviews)24. Two people (ISM, IB) considered each publication independently and decided on the methodological quality through discussions until consensus.\n\nThe final decision on which reviews to include was done through agreement between two of the authors (ISM and AA). Table 1 contains documentation on characteristics of the included reviews, including methodological quality.\n\nDue to overlap of treatment comparisons for the same population, we included the review with the newest search (and completeness of this search by considering the included primary studies) and the best quality.\n\nISM extracted data from the systematic reviews and AA checked its accuracy. As this was an overview of systematic reviews, we extracted information as it was reported in the systematic reviews, including any supplementary tables or appendixes. We did not retrieve primary studies to provide additional information about interventions or results.\n\nFrom the systematic reviews, we extracted information about the primary studies populations, characteristics of the interventions and comparison groups, duration of the interventions, follow-up periods, outcome measures and pooled effect estimates for each outcome. In cases were the effect estimates were not pooled in a meta-analysis, we reported the results of each individual study for each outcome.\n\nWe did not attempt any reanalysis, but present results as reported in the systematic reviews. For reviews also including studies on adult populations, we only extracted information from studies of children and adolescents. When reported, the effect estimates were presented with relevant measures of uncertainty.\n\nWe assessed our confidence in the evidence of effect for each outcomes using the GRADE methodology (the Grading of Recommendations Assessment, Development and Evaluation)25. If the systematic review authors had already completed a GRADE assessment, we reviewed this. We describe our confidence in the effect estimates as high, moderate, low or very low for each outcome.\n\n\nResults\n\nAll 1259 references in the INSUM database was reviewed for potential relevance (see Figure 1). Additionally, we also identified 12 records through hand-searches. We excluded 1242 of these based on title or summary, mainly because they focused on other diagnosis or problem-areas than self-harm and/or suicide. Overall, 29 full texts were retrieved, 12 were excluded because they did not fulfil the inclusion criteria. Out of 18 potentially included reviews, 9 were excluded because of overlap (see Table 2 for excluded studies).\n\nFigure 1 describes the search-process and the number of articles excluded in each step. Eight systematic reviews1,13,14,26–30, including summary of new evidence of two of them31,32, were consequently included in the analysis. One review was identified after we had completed the analysis33 and is therefore not included in the present review of systematic reviews.\n\nAlthough the initial cut-off for age in our population was 18, two of the reviews included studies with young people up to 2426,27. These were included because the upper age limit used to define adolescence in research on self-harm and suicides varies between 18 and 255.\n\nThe eight included systematic reviews1,13,14,26–32 were assessed for quality (see Table 1). Overall, the reviews were of high methodological quality, even though some of the reviews lacked a priori design, systematic searches for grey literature and assessment of publication bias. We appraised three systematic reviews14,27,30 with AMSTAR-scores in the range of 6–8, and the remaining five1,13,26–29,31,32 with AMSTAR-scores in the range of 9–11.\n\nThe reviews included a broad range of interventions. Most of the studies included adolescent populations in the age-range 12 to 18, with some exceptions of samples including younger children or young adults up to the age of 24. Preventive interventions were either primary prevention strategies for mixed population based samples (suicide awareness campaigns and other school-based prevention programs, screening for suicide risk) or secondary preventions strategies (local approaches following suicide clusters, suicide prevention in residential custodial and detention settings, interventions to support children and adolescents bereaved or affected by a suspected suicide)14,26,27. The reviews also included psychosocial or psychological intervention in cases of existing self-harm (defined as a history of at least one episode of self-harm) (therapeutic assessment, mentalization based therapy, dialectic behaviour therapy, cognitive behaviour therapy, developmental group therapy, compliance enhancement, home-based family intervention, emergency green cards, digital interventions for self-management of suicidal ideation and self-harm, postcards)13,27,28,31.\n\nThe effects of interventions are presented by type population (young people with or without an identified risk, or with existing self-harm, e.g. a history of at least one episode of self-harm) and by treatment comparison. Our assessment of certainty on the evidence corresponds to GRADE-tables in Table 3–Table 18. For comparisons with many outcomes, we report the main outcomes in the present results section. See GRADE-Table 3–Table 18 for the remaining outcomes.\n\n1. Downgraded by 1 level due to unclear risk of bias.\n\n2. Downgraded by 2 levels because of study design (observational study).\n\n3. Downgraded by 1 level due to imprecision (only 1 study).\n\n4. Downgraded by 1 level due to imprecision (few incidences).\n\n5. Downgraded by 1 level due to lack of reporting (effect estimates and measure of uncertainty)\n\n6. Downgraded -1 due to heterogeneity.\n\nFor the following interventions (versus treatment as usual (TAU) or alternative interventions), the review authors also searched for research on effects, but did not identify studies on children and adolescents under the age of 18 were not identified. These are primary and secondary preventive interventions (reducing access to means, local suicide plans, local media reporting of suicides in newspapers, Internet or other digital channels, suicide prevention in residential custodial and detention settings)26 and interventions for existing self-harm (assessment in children and adolescents at the emergency department, psychoeducation, pharmacological treatment or a combination of pharmacological treatment and psychotherapy, nutrition, other psychotherapeutic approaches such as problem-solving therapy, psychodynamic therapy, multi-systemic therapy, supportive therapy, or other psychosocial approaches such as counselling, self-management, respite care, assertive outreach)1,28–32.\n\nSchool-based suicide prevention programs versus TAU, alternative interventions, wait list or no intervention. The evidence includes 13 studies with <337 221 children and adolescents aged 10 to 23, as well as personnel in different local arenas working with young people14,26. In one of the studies, the participants (n=320 500) were habitants in a county in which county-based prevention programs were implemented. These participants included school students and personnel in schools and other local arenas. School-based prevention programs probably reduce suicidal ideation (RR 0.67, 95% KI 0.48 to 0.93, moderate certainty⊕⊕⊕⊖) and suicide attempts (RR 0.53, 95% KI 0.36 to 0.80, moderate certainty⊕⊕⊕⊖) at three to 12 months. Regarding suicide attempts, three studies conclude accordingly at six- and 12-month follow-up period. This effect possibly holds at ≥two- and 15-year follow-up (low certainty⊕⊕⊖⊖). Further, school-based interventions possibly reduce the rate of completed suicides at three-year follow-up (low certainty⊕⊕⊖⊖). Effects on help-seeking and possible unwanted effects are unclear since the evidence for these outcomes is of very low certainty⊕⊖⊖⊖. See Table 3.\n\nPrimary prevention: local approaches following suicide clusters versus historical control. The evidence includes three studies with children and adolescents between the ages of 10 and 2426. Follow-up period was up to four years. The evidence of effects of local approaches following suicide clusters is of very low certainty⊕⊖⊖⊖. See Table 4.\n\n1. Downgraded by 2 due to study design (observational studies).\n\n2. Downgraded by 1 due to lack of precision (few incidences/short follow-up period).\n\nSecondary prevention: interventions to support children and adolescents bereaved or affected by a suspected suicide compared to TAU or historical control. The evidence includes two studies26. The evidence of effects of interventions to support children and adolescents bereaved or affected by a suspected suicide is of very low certainty⊕⊖⊖⊖. See Table 5.\n\n1. Downgraded by 1 level due to risk of bias (no blinding).\n\n2. Downgraded by 1 level due to imprecision (few participants).\n\n3. Downgraded by 1 level due to imprecision (only 1 study).\n\nPrimary prevention: screening for suicide risk versus no screening. The evidence is based on one review27. The review authors did not identify studies evaluating beneficial effects of screening as a preventive strategy in children or adolescents. They did however identify two studies evaluating harms associated with screening for psychological distress and a history of deliberate self-harm and suicidal ideation in primary care settings. The studies comprised of 2650 adolescents between 13 and 19 years old, and the evidence is of very low certainty⊕⊖⊖⊖. See Table 6.\n\n1. Downgraded by 1 level due to unclear risk of bias (not reported).\n\n2. Downgraded by 1 level due to imprecision (few incidences).\n\n3. Downgraded by 1 level due to lack of reporting of numbers.\n\n4. Downgraded by 2 levels due to not reported study design.\n\nInterventions for existing self-harm: therapeutic assessment versus TAU. The evidence includes one study with 70 adolescents, 12 to 18-year olds, referred for a psychosocial assessment following an episode of self-injury or self-poisoning, irrespective of intent28. Length of intervention was one hour and 40 minutes. Follow up was 12 and 24 months. The evidence of effects of therapeutic assessment is of very low certainty⊕⊖⊖⊖. See Table 7.\n\n1. Downgraded by 1 level due to risk of bias (no blinding).\n\n2. Downgraded by 1 level due to imprecision (few participants).\n\n3. Downgraded by 1 level due to imprecision (only 1 study).\n\nInterventions for existing self-harm: mentalization based therapy (MBT-A) versus TAU. The evidence includes one study with 80 adolescents, 12 to 17-year olds, diagnosed with depression presenting to emergency departments or community psychiatric services following an episode of self-injury or self-poisoning, irrespective of whether suicidal intent was present28. Length of treatment was 12 months, and follow-up period was also 12 months. The evidence of effects of therapeutic assessment is of very low certainty⊕⊖⊖⊖. See Table 8.\n\n1. Downgraded by 1 level due to risk of bias (no blinding).\n\n2. Downgraded by 1 level due to imprecision (few participants/incidences).\n\n3. Downgraded by 1 level due to imprecision (only 1 study).\n\nInterventions for existing self-harm: dialectical behaviour therapy (DBT-A) versus TAU or enhanced TAU. The evidence includes two studies with 106 adolescents between the age of 12 and 19 years old with a history of multiple episodes self-harm28,31. Length of treatment was 19 weeks. Follow-up period was 16 weeks and six months. Based on the available evidence DBT-A has little or no effect on repetition or frequency of self-harm (OR 0.72, 95% KI 0.12 to 4.40, low certainty⊕⊕⊖⊖). DBT-A may have a moderate effect on reduction of suicidal ideation (SMD -0.62, 95% KI -1.07 to -0.16, low certainty⊕⊕⊖⊖). The certainty of the evidence for other outcomes is very low⊕⊖⊖⊖. See Table 9.\n\n1. Downgraded by 1 level due to risk of bias.\n\n2. Downgraded by 1 level due to imprecision (few participants).\n\n3. Downgraded by 1 level due to heterogeneity.\n\n4. Downgraded by 1 level due to imprecision (very wide confidence interval).\n\n5. Downgraded by 1 level due to imprecision (only 1 study).\n\n6. Downgraded by 1 level due to imprecision (few incidences).\n\nInterventions for existing self-harm: cognitive behaviour therapy (CBT) versus non-directive psychotherapy. The evidence contains one study with 39 adolescents between the age of 12 and 17 presenting to a paediatric general or psychiatric facility following self-injury in which an intent to die was indicated28. Length of treatment was six months. Follow-up period was three, six and 12 months. The certainty of the evidence for CBT versus non-directive psychotherapy is very low⊕⊖⊖⊖. See Table 10.\n\n1. Downgraded by 2 levels due to serious risk of bias.\n\n2. Downgraded by 1 level due to conflict of interest.\n\n3. Downgraded by 1 level due to imprecision (only 1 study).\n\n4. Downgraded by 1 level due to imprecision (few participants/incidences).\n\nInterventions for existing self-harm: developmental group therapy versus TAU. The evidence contains three studies of 487 adolescents, 12 to 17-year olds, referred to child and adolescent services following an episode of intentional self-injury or self-poisoning, irrespective of intent28. Acute treatment phase was six weekly sessions, followed by weekly or biweekly booster sessions for as long as required. Follow-up period was between six and 12 months. Based on the available evidence, the effects of developmental group therapy are uncertain on the following outcomes: repetition of self-harm (six months: OR 1.72 95% KI 0.56-5.24, 12 months: OR 0.80 95% KI 0.22 to 2.97), depression (six months: MD 0.40 95% KI -2.76 to 3.55, 12 months: MD -0.93 95% KI -4.03 to 2.17), suicidal ideation (six months: MD 1.27 95% KI -7.74 to 10.28, 12 months: MD -1.51 95% KI 9.62 to 6.59) or suicide (no suicides). The evidence for all the outcomes is of low certainty⊕⊕⊖⊖. See Table 11.\n\n1. Downgraded by 1 level due to risk of bias (lack of blinding).\n\n2. Downgraded by 1 level due to imprecision (wide confidence interval).\n\n3. Downgraded by 1 level due to imprecision (few incidences).\n\nInterventions for existing self-harm: compliance enhancement versus TAU. The evidence contains one study of 76 adolescents, 12 to 19-year olds, admitted to the emergency department of a general hospital following an episode of self-injury, irrespective of intent, and/or with an increased risk for suicidality28. Length of treatment was eight weeks. Follow-up period was three months. The evidence of effects of compliance enhancement is of very low certainty⊕⊖⊖⊖. See Table 12.\n\n1. Downgraded by 1 level due to imprecision (only 1 study).\n\n2. Downgraded by 1 level due to imprecision (few participants).\n\n3. Downgraded by 2 levels due to serious risk of bias.\n\n4. Downgraded by 1 level due to unclear risk of bias.\n\nInterventions for existing self-harm: home based family intervention versus TAU. The evidence contains one study in a sample of adolescents aged 16 years or younger referred to child and adolescent mental health services following an episode of self-poisoning irrespective of intent28. The intervention was a manualised home-based family therapy intervention. Follow-up period was six months. The evidence of effects of home-based family intervention is of very low certainty⊕⊖⊖⊖. See Table 13.\n\n1. Downgraded by 1 level due to risk of bias (lack of blinding).\n\n2. Downgraded by 1 level due to imprecision (only 1 study).\n\n3. Downgraded by 1 level due to imprecision (few participants/incidences).\n\nInterventions for existing self-harm: emergency green cards plus TAU versus TAU. The evidence contains one study with 105 adolescents between the ages of 12 and 16 who were admitted to hospital following an episode of self-injury or self-poisoning28. The intervention was emergency green cards in addition to usual care. The green card acted as a passport to re-admission into a paediatric ward at the local hospital. Length of treatment was 12 months. Follow-up period was 12 months. The evidence of effects of emergency green cards is of very low certainty⊕⊖⊖⊖. See Table 14.\n\n1. Downgraded by 2 levels due to serious risk of bias.\n\n2. Downgraded by 1 level due to imprecision (only 1 study).\n\n3. Downgraded by 1 level due to imprecision (few participants).\n\nInterventions for existing self-harm: digital interventions for self-management of suicidal ideation and self-harm versus psychoeducation or historical control. The evidence contains three studies with 184 adolescents reporting suicidal thoughts and/or receiving treatment for depression13. The interventions spanned from two to 12 weeks and follow-up was post treatment. The evidence of effects of digital interventions for self-management is of very low certainty⊕⊖⊖⊖. See Table 15.\n\n1. Downgraded by 1 level due to risk of bias.\n\n2. Downgraded by 1 level due to imprecision (few participants).\n\n3. Downgraded by 2 levels due to study design (2 out of 3 studies were observational).\n\nInterventions for existing self-harm: postcards versus TAU. The evidence is based on two systematic reviews27,31. One of the reviews31 included one study with 2300 adolescents and young adults over the age of 12 previously admitted to a specialist poisons hospital after self-poisoning. The other review27 included one study of 165 adolescents and young adults of 15 to 24 years old with a history of suicidal threats, ideation, attempts and/or self-injury who did not meet entry criteria for service because they either were not unwell enough or were receiving treatment elsewhere. Follow-up was post study. The evidence of effects of postcards is of very low certainty⊕⊖⊖⊖. See Table 16.\n\n1. Downgraded by 1 level due to possible lack of generalizability (Study 2 is an adolescent population in Teheran).\n\n2. Downgraded by 1 level due to unclear risk of bias.\n\n3. Downgraded by 1 level due to lack of reporting effect estimates and measurement of uncertainty.\n\n4. Downgraded by 1 level due to imprecision (only 1 study).\n\n\nDiscussion\n\nThe major contribution of this review is to provide children, adolescents and their families, clinicians and researchers with an overview of research regarding the effects of interventions for young people to prevent suicide and (re)occurrence of self-harm. For this purpose, we have used systematic and transparent criteria23–25. The results of our review should be supplemented with other relevant research and integrated with clinical expertise as well as the child’s or adolescent’s and their caregiver’s values and preferences34,35.\n\nA limitation of overviews of reviews, and consequently of this present report, is that the analyses are based on secondary reporting and the interpretation of the review authors. Thus, the primary studies may have provided more information than what is reported in the reviews we included. Nevertheless, the present report provides insight into the certainty of the evidence of effects of treatments and other interventions that have been evaluated. This report also identifies important research gaps for interventions where no studies have been conducted. Acknowledging that the effects of these interventions in reducing self-harm and suicide are uncertain can prompt new research efforts important for children and adolescents.\n\nIt is also worth noting that the present report only included reviews of studies where the population was children and young people with existing self-harm or preventive strategies for children and adolescents with or without an identified risk of self-harm and suicide. As mentioned in the introduction, self-harm and suicide are outcomes associated with other underlying difficulties. Therefore, evidence from studies including young people with problem such as other mental health issues typically associated with self-harm may provide important direction in decision-making when faced with self-harm and suicide. However, in the existing research-base on e.g. psychosis, depression and anxiety, self-harm and suicide are rarely investigated as outcomes36–38. According to the existing low certainty evidence, combination treatment for depression (pharmacological treatment plus psychotherapy) may lead to a reduced risk for suicide37.\n\nBased on the available research, there is moderate certainty evidence that school-based interventions can prevent suicidal ideation and suicide attempts short term, and low certainty evidence that they can prevent suicide attempts long term.\n\nThe certainty of the evidence for the effects of screening children and young people for symptoms of depression and a history of self-harm or suicidal ideation in the general population is very low, and the benefits and harms of such interventions are therefore unknown.\n\nLocal suicide plans are a recommended strategy in some countries26,39. However, the effects of such plans on preventing self-harm and suicide in children and young people is yet to be evaluated in research.\n\nWe identified no studies evaluating the effects of reducing access to means from children and young people specifically. However, studies on the general population, including populations with adults, suggests that this may be an effective strategy26.\n\nFurthermore, there is a need for more research on how media reporting of suicides affects suicide rates in children and young people. However, studies at a population level suggests that certain forms of media reporting are associated with an increase in suicides26. Guidelines on how to report on suicides is one suggested strategy to address the harms of such reporting26.\n\nThe certainty of evidence for community-based interventions following suicide clusters is very low. The best strategies for addressing this phenomenon and later suicides following suicide clusters are therefore unknown. Even though research is scarce, some recommendations are agreed upon, e.g. provision of information to relevant agencies in the community and providing support for those directly affected or other vulnerable individuals40.\n\nThe reviews we identified also searched for studies targeting young people in residential custodial and detention settings. No studies evaluating interventions to prevent suicide in this high-risk population were identified. Therefore, effects uncertain.\n\nAnother high-risk group is young people bereaved or affected by a suicide in their family or other network. Two studies were identified addressing the effects of support-interventions in this population. However, the evidence is of very low certainty.\n\nBased on the available evidence, it is uncertain which approach to risk assessment of young people after an episode of self-harm is most appropriate. Furthermore, the effects of psychoeducation, psychological therapy, psychosocial interventions, digital interventions for self-management and nutrition for treating young people with existing self-harm are uncertain. For most of these interventions no studies were found, or the certainty of the evidence was very low.\n\nTwo treatment comparisons evaluating psychological therapy provided evidence of their effectiveness (low certainty); dialectical behavioural therapy and developmental group therapy. Both treatments were compared to alternative psychological therapy, and there was little or no important difference in effect on repetition of self-harm compared to alternative follow up. However, of notice, there was substantially higher (although not statistically significant) repetition of self-harm amongst adolescents participating in group developmental therapy compared to those receiving individual therapy at six-month follow-up. At 12-month follow-up, there was little or no important effect on self-harm.\n\nWe found no studies on direct comparisons of pharmacological treatments or on the effects of combination therapy (pharmacological plus psychotherapy).\n\nThe evidence of effects of organization of services, such as home-based treatment and use of emergency green cards, is of very low certainty.\n\nSuicide clusters, although rare, is of major concern. When faced with this phenomenon or in fear of potential social contagion following the suicide of an individual, communities are expected to act to prevent further social contagion and clustering.\n\n\nConclusions\n\nOverall, evidence of moderate to low certainty suggests that school-based suicide prevention programs can prevent suicide and suicide attempts in young people.\n\nThe effects of community-based interventions following suicide clusters and local suicide plans are uncertain. Furthermore, it is not possible to make any conclusions about the benefits and harms of screening in young people or and without known risk of self-harm and suicide.\n\nEvidence of low certainty suggests that dialectical behavioural therapy and developmental group therapy are equally as effective on repetition of self-harm as enhanced treatment as usual (individual and/or family psychotherapy). The effects of evidence for other interventions preventing self-harm and suicide is of very low certainty or remains to be evaluated. These includes approaches to risk assessment and how to best organize the care of young people with known self-harm or suicide risk.\n\nOur review suggests that preventive strategies can reduce suicide risk. However, there is a lack of research on effects of recommended practices, such as local suicide plans and approaches to risk assessment. Screening for suicide risk as primary prevention may provide the opportunity of early detection, and if precise, offers the opportunity to provide young people at risk with appropriate treatment. However, screening is resource demanding, and beneficial and possible harmful effects are uncertain. When implemented, local suicide plans, approaches to risk assessment and screening programs should be closely evaluated.\n\nIt is recommended that communities prepare for situations with a risk for social contagion and suicide clusters. Research evaluating strategies to prevent clustering of suicides is scarce, and the studies we found used inappropriate designs to capture the potential beneficial or harmful effects of these interventions. We suggest that researchers design appropriate observational studies, allowing for enough observations pre- and post-implementation of preventive measures to inform policy.\n\nThere is great uncertainty associated with the effects of treatment strategies for young people with existing self-harm. More research is needed, including on younger children and long-term follow up.\n\nSelf-harm is a common reason for referral of adolescents in child and adolescent psychiatric services, and often accompanies other psychiatric symptoms presented in such settings. It follows that psychological or psychosocial approaches showing promise in treatment and prevention of conditions associated with self-harm and/or suicidality, such as depression and psychosis, should be considered in treatment of repeated self-harm. In general, when effects of interventions preventing self-harm and suicide in children and adolescents are uncertain due to lack of research or evidence of very low certainty, policy makers and health providers should consider evidence from population-based studies and adults.\n\nIt is crucial to be mindful that our own preventive actions or treatment efforts possibly could contribute to an increased risk for self-harm and suicide. Practice should be evaluated, and researchers should investigate harmful effects as well as beneficial effects of interventions.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: Appendix 1 search strategy. https://doi.org/10.6084/m9.figshare.822384222\n\nThis project contains the following extended data:\n\nAppendix_1_Search_strategy.pdf (Study search strategy)\n\nFigshare: PRISMA checklist for ‘The effects of interventions preventing self-harm and suicide in children and adolescents: an overview of systematic reviews’. https://doi.org/10.6084/m9.figshare.8223863.v141\n\nFigshare: PRISMA flow chart for ‘The effects of interventions preventing self-harm and suicide in children and adolescents: an overview of systematic reviews’. https://doi.org/10.6084/m9.figshare.8223875.v142\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThe present paper was funded by the Regional Centre for Child and Adolescent Mental Health, Eastern and Southern Norway (RBUP), the Regional Centre on violence, trauma and suicide prevention, Eastern Norway and the Norwegian Directorate of Health.\n\n\nAcknowledgements\n\nWe would like to thank our colleagues at the Regional Centre for Child and Adolescent Mental Health, Eastern and Southern Norway, Ingrid Borren and Karianne Thune Hammerstrøm, for respectively assessing methodological quality of publications and reviewing publications indexed in IN SUM.\n\n\nReferences\n\nNICE: Self-harm in over 8s: Long-term management. Clinical guideline CG133 [Internet]. London: National Institute for Excellence and Health (NICE); 2011. Reference Source\n\nHawton K, Hall S, Simkin S: Deliberate self-harm in adolescents: a study of characteristics and trends in Oxford, 1990-2000. J Child Psychol Psychiatry. 2003; 44(8): 1191–98. PubMed Abstract \| Publisher Full Text\n\nEdmondson AJ, Brennan CA, House AO: Non-suicidal reasons for self-harm: A systematic review of self-reported accounts. J Affect Disord. 2016; 191: 109–17. PubMed Abstract \| Publisher Full Text\n\nNock MK, Borges G, Bromet EJ, et al.: Suicide and suicidal behavior. Epidemiol Rev. 2008; 30(1): 133–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHawton K, Saunders KE, O’Connor RC: Self-harm and suicide in adolescents. Lancet. 2012; 379(9834): 2373–82. PubMed Abstract \| Publisher Full Text\n\nBertolote JM, Fleischmann A: Suicide and psychiatric diagnosis: a worldwide perspective. World Psychiatry. 2002; 1(3): 181–85. PubMed Abstract \| Free Full Text\n\nMuehlenkamp JJ, Claes L, Havertape L, et al.: International prevalence of adolescent non-suicidal self-injury and deliberate self-harm. Child Adolesc Psychiatry Ment Health. 2012; 6: 10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMohl B, Skandsen A: The prevalence and distribution of self-harm among Danish high school students. Personality and Mental Health. 2012; 6(2): 147–55. Publisher Full Text\n\nJoiner TE Jr, Conwell Y, Fitzpatrick KK, et al.: Four studies on how past and current suicidality relate even when \"everything but the kitchen sink\" is covaried. J Abnorm Psychol. 2005; 114(2): 291–303. PubMed Abstract \| Publisher Full Text\n\nSpirito A, Esposito-Smythers C: Attempted and completed suicide in adolescence. Annu Rev Clin Psychol. 2006; 2: 237–66. PubMed Abstract \| Publisher Full Text\n\nLilley R, Owens D, Horrocks J, et al.: Hospital care and repetition following self-harm: multicentre comparison of self-poisoning and self-injury. Br J Psychiatry. 2008; 192(6): 440–5. PubMed Abstract \| Publisher Full Text\n\nWasserman D, Cheng Q, Jiang GX: Global suicide rates among young people aged 15-19. World Psychiatry. 2005; 4(2): 114–20. PubMed Abstract \| Free Full Text\n\nWitt K, Spittal MJ, Carter G, et al.: Effectiveness of online and mobile telephone applications ('apps') for the self-management of suicidal ideation and self-harm: a systematic review and meta-analysis. BMC Psychiatry. 2017; 17(1): 297. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSBU: Skolebaserade program för att förebygga sjëlvskadebeteende inklusive suicidforsök [Internet]. Stocholm: Swedish agency for health techonogy assessment and assessment of social services (SBU); 2015. Reference Source\n\nCusimano MD, Sameem M: The effectiveness of middle and high school-based suicide prevention programmes for adolescents: a systematic review. Inj Prev. 2011; 17(1): 43–9. PubMed Abstract \| Publisher Full Text\n\nFrey LM, Hunt QA: Treatment For Suicidal Thoughts and Behavior: A Review of Family-Based Interventions. J Marital Fam Ther. 2017; 44(1): 107–124. PubMed Abstract \| Publisher Full Text\n\nOugrin D, Latif S: Specific psychological treatment versus treatment as usual in adolescents with self-harm: systematic review and meta-analysis. Crisis. 2011; 32(2): 74–80. PubMed Abstract \| Publisher Full Text\n\nWei Y, Kutcher S, LeBlanc JC: Hot Idea or Hot Air: A Systematic Review of Evidence for Two Widely Marketed Youth Suicide Prevention Programs and Recommendations for Implementation. J Can Acad Child Adolesc Psychiatry. 2015; 24(1): 5–16. PubMed Abstract \| Free Full Text\n\nIoannidis JP, Greenland S, Hlatky MA, et al.: Increasing value and reducing waste in research design, conduct, and analysis. Lancet. 2014; 383(9912): 166–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDARE: Database of abstracts of reviews of effects [Internet]. York: University of York. Center for reviews and dissemination. [retrieved 28.08.18]. Reference Source\n\nIN SUM: a database of systematic reviews on effects of child mental health and welfare interventions [Internet]. Oslo: Regional Centre for Child and Adolescent Mental Health, Eastern and Southern Norway (RBUP). [retrieved 28.08.18]. Reference Source\n\nMorken IS: “Appendix 1 Search Strategy”. figshare. 2019. http://www.doi.org/10.6084/m9.figshare.8223842.v1\n\nHiggins JPT, Green S (editors): Cochrane Handbook for Systematic reviews of Interventions Version 5.1.0 [Internet]. The Cochrane Collaboration; 2011. Reference Source\n\nAMSTAR: assessing the methodological quality of systematic reviews [Internet]. [retrieved 28.08.18]. Reference Source\n\nGRADE: grading of recommendations assessment, development and evaluation [Internet]. [retrieved 28.08.18]. Reference Source\n\nNICE: Preventing suicide in community and custodial settings [Internet]. London: National Institute for Excellence and Health (NICE); 2018. Reference Source\n\nO’Connor E, Gaynes BN, Burda BU, et al.: Screening for and treatment of suicide risk relevant to primary care: a systematic review for the U.S. Preventive Services Task Force. Ann Intern Med. 2013; 158(10): 741–54. PubMed Abstract \| Publisher Full Text\n\nHawton K, Witt KG, Taylor Salisbury TL, et al.: Interventions for self-harm in children and adolescents. Cochrane Database Syst Rev. 2015; (12): CD012013. PubMed Abstract \| Publisher Full Text\n\nNICE: Self-harm in over 8s: short-term management and prevention of recurrence. Clinical guideline CG16 [Internet]. London: National Institute for Excellence and Health (NICE); 2004. Reference Source\n\nOugrin D, Tranah T, Stahl D, et al.: Therapeutic interventions for suicide attempts and self-harm in adolescents: systematic review and meta-analysis. J Am Acad Child Adolesc Psychiatry. 2015; 54(2): 97–107.e2. PubMed Abstract \| Publisher Full Text\n\nNICE: Appendix A.2: summary of new evidence from 4-year surveillance of Self-harm in over 8s: long-term management (2011) NICE guideline CG133 [Internet]. London: National Institute for Excellence and Health (NICE); 2016. Reference Source\n\nNICE: Appendix A.1: summary of new evidence from 12-year surveillance of self-harm in over 8s: short term management and prevention of recurrence (2004) NICE guideline CG16 [Internet]. London: National Institute for Excellence and Health (NICE); 2016. Reference Source\n\nMcCabe R, Garside R, Backhouse A, et al.: Effectiveness of brief psychological interventions for suicidal presentations: a systematic review. BMJ Psychiatry. 2018; 18(1): 120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDawes M, Summerskill W, Glasziou P, et al.: Sicily statement on evidence-based practice. BMC Med Educ. 2005; 5(1): 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAPA Presidential Task Force on Evidence Based Practice. Am Psychol. 2006; 61(4): 271–85. Publisher Full Text\n\nDahlgren A, Hammerstrøm K, Bjørndal A, et al.: Kunnskapsoppsummering: Effekt av tiltak for angstlidelser hos barn og unge [Internet]. Oslo: Regional Centre for Child and Adolescent Mental Health, Eastern and Southern Norway; 2018. Reference Source\n\nDahlgren A, Hammerstrøm K, Bjørndal A, et al.: Kunnskapsoppsummering: Effekt av tiltak ved depresjon hos barn og unge [Internet]. Oslo: Regional Centre for Child and Adolescent Mental Health, Eastern and Southern Norway; 2018. Reference Source\n\nDahlgren A, Morken IS, Karlsen K, et al.: Kunnskapsoppsummering: Effekt av tiltak ved psykoselidelser hos barn og unge [Internet]. Oslo: Regional Centre for Child and Adolescent Mental Health, Eastern and Southern Norway; 2018. Reference Source\n\nNorwegian Directorate of Health: Veiledende materiell for kommunene om forebygging av selvskade og selvmord [Internet]. Oslo: The Norwegian Directorate of Health; 2017. Reference Source\n\nPublic Health England: Identifying and responding to suicide clusters and contagion. A practice resource [Internet]. London: Public Health England; 2015. Reference Source\n\nMorken IS: “PRISMA Checklist”. figshare. 2019. http://www.doi.org/10.6084/m9.figshare.8223863.v1\n\nMorken IS: “PRISMA Flow Chart of Search Strategy”. figshare. 2019. http://www.doi.org/10.6084/m9.figshare.8223875.v1" }	[ { "id": "52990", "date": "11 Nov 2019", "name": "Sze Ngar Vanessa Yuan", "expertise": [ "Reviewer Expertise Clinical psychology", "mainly child and adolescent mental health", "specifically self-harm or autism." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article evaluates the effects of self-harm and suicide prevention interventions for children and adolescents. The rationale behind the study is sufficiently supported by different research. The methodology provided is sufficient except it is unclear why only systematic reviews published in 2012 or later are included. It is also unclear what search terms were used for future replications.\nThe authors report a range of outcome measures in the results that may not be directly related to self-harm. Such outcomes (e.g. treatment engagement) were not further commented on or summarised. Overall, the systematic review is very comprehensive but the discussion and summary could be more coherent and with a better flow. For instance, the authors can attempt to comment on the implications of TAU control group versus other active intervention control groups.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "5216", "date": "18 Feb 2020", "name": "Ida Sund Morken", "role": "Author Response", "response": "1) The methodology provided is sufficient except it is unclear why only systematic reviews published in 2012 or later are included. · We thank reviewer 1 for pointing out that this was unclear. This cut-off is pragmatic but similar to that practiced by others, for example, the Cochrane Library (https://community.cochrane.org/editorial-and-publishing-policy-resource/cochrane-review-development/cochrane-review-updates), in considering that an older review is obsolete and no longer a reliable basis for evidence and in need for being updated. It takes time before a review is published, consequently a review may be published one to three years later than the search was done for primary studies. This has improved with time and new publication standards for reviews. Thus, a review published earlier than 2012 may not include primary studies published the last >10 years. A sentence to explain this has now been added to the manuscript. 2) It is also unclear what search terms were used for future replications.· The literature search for this review was completed in August 2018 and is largely based on IN SUM: a database of systematic reviews on effects of child mental health and welfare interventions. We reviewed all references indexed in IN SUM. IN SUM indexes reviews related to children’s and young people’s mental health from the following databases: Cochrane Database of Systematic Reviews, Campbell Library, PsycINFO, MEDLINE, Embase, Web of Science, Database of Abstracts of Reviews of Effects (DARE) and Evidence Based Mental Health. A description of IN SUMs searching strategy is included in extended data. We have now made it explicit that the search-words are included in the IN SUM Search Strategy (extended data, reference 22), we provide examples of search words, and we point out that we screened all the references in IN SUM. 3) The authors report a range of outcome measures in the results that may not be directly related to self-harm. Such outcomes (e.g. treatment engagement) were not further commented on or summarised. · Thank you for pointing this out, as research on other outcomes are often highly relevant. For this review we included all outcomes as reported by the review authors. Effect estimates and judgements of certainty for each such outcome is reported for all pooled estimates. However, in line with the GRADE recommendations, we only make conclusions on outcomes judged to be of low, moderate or high certainty. When evidence is of very low certainty, the effects of these outcomes are considered to be too uncertain as to make any conclusions. 4) Overall, the systematic review is very comprehensive but the discussion and summary could be more coherent and with a better flow. For instance, the authors can attempt to comment on the implications of TAU control group versus other active intervention control groups.· Thank you for this feedback. We have now tried to make the discussion and summary more coherent, see introduction, discussion and summary. We have also commented on the implications of TAU control group versus other active intervention control groups, see “Effects of interventions for existing self-harm: summary of findings and implications”." } ] }, { "id": "58596", "date": "15 Jan 2020", "name": "Gianluca Serafini", "expertise": [ "Reviewer Expertise Psychopathology and neurobiology of suicidal behavior and major affective disorders." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you very much for asking me to review the present manuscript.\nThis is, in summary, an interesting paper aimed to evaluate the effects of interventions preventing self-harm and suicide in children and adolescents in an overview of systematic reviews. The authors reported that (moderate certainty evidence) school-based interventions prevent suicidal ideation and attempts short term, and possibly with long term effects on suicide attempts. Overall, the effects of community-based interventions following suicide clusters and local suicide plans resulted uncertain, as are the benefits and harms of screening young people for suicide risk. In addition, the effects of most interventions targeting children and adolescents with known self-harm were uncertain. They added that (low certainty evidence) dialectical behavioural therapy and developmental group therapy are equally as effective on repetition of self-harm as enhanced treatment as usual.\nThe authors may find as follows my main comments/suggestions:\nFirst, as the authors, throughout the Introduction section, correctly stated that self-harm and suicide are associated with relevant psychosocial impairment and result from underlying factors such as other mental health problems, exposure to traumatic events or other difficult circumstances in the young person’s environment, they might even mention that the emotional turmoil in the case of suicide survivors of patients died by suicide may last a long time, and in some cases, may end with their own suicide. Thus, together with self-harm and suicide, it is fundamental to understand the bereavement process after the suicide of a significant other to provide a proper care, reduce stigma, and improve the outcomes. In addition, specific biological factors such as prolactin and thyroid hormone levels may be dysregulated and significantly associated with self-harm and suicide attempts and even involved in a complex compensatory mechanism to correct reduced central serotonin activity. The assumption that prolactin and thyroid hormones may be associated or even predict a suicide attempt is of great importance given the availability of such data in everyday clinical practice. Physicians of any kind as well as mental health professionals should be aware of the importance to insert as much information possible in the assessment of suicide and self-harm risk. Thus, given the above mentioned information, the authors could include throughout the manuscript, some published papers regarding the mentioned topics (PMID: 24082246; 31091772; 28843902; 22748186; 12866334)12345.\nIn addition, why the authors decided to include all publications in English, Norwegian, Danish or Swedish rather than simply including only studies in English language is a matter of debate and needs to be specified.\nMoreover, the authors should immediately present and discuss, in the first lines of the Discussion section, their most relevant study findings. Conversely, they seem to focus with redundancy on the main aims/objectives of the paper which have been already presented elsewhere.\nAlthough the authors reported that the present analyses are based on secondary reporting and the interpretation of the review authors as well as that the present report included only reviews of studies where the population was children and young people with existing self-harm, the most relevant limitations/shortcomings of the present study need to be more carefully described for the general readership.\nFinally, what is the take-home message of this manuscript? While the authors stated that practice should be evaluated, and researchers should investigate harmful effects as well as beneficial effects of interventions, they failed, in my opinion, to provide some conclusive remarks about their findings. Here, some further details/information are needed.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "5217", "date": "18 Feb 2020", "name": "Ida Sund Morken", "role": "Author Response", "response": "1) First, as the authors, throughout the Introduction section, correctly stated that self-harm and suicide are associated with relevant psychosocial impairment and result from underlying factors such as other mental health problems, exposure to traumatic events or other difficult circumstances in the young person’s environment, they might even mention that the emotional turmoil in the case of suicide survivors of patients died by suicide may last a long time, and in some cases, may end with their own suicide. Thus, together with self-harm and suicide, it is fundamental to understand the bereavement process after the suicide of a significant other to provide a proper care, reduce stigma, and improve the outcomes. In addition, specific biological factors such as prolactin and thyroid hormone levels may be dysregulated and significantly associated with self-harm and suicide attempts and even involved in a complex compensatory mechanism to correct reduced central serotonin activity. The assumption that prolactin and thyroid hormones may be associated or even predict a suicide attempt is of great importance given the availability of such data in everyday clinical practice. Physicians of any kind as well as mental health professionals should be aware of the importance to insert as much information possible in the assessment of suicide and self-harm risk. Thus, given the above mentioned information, the authors could include throughout the manuscript, some published papers regarding the mentioned topics (PMID: 24082246; 31091772; 28843902; 22748186; 12866334). Thank you for interesting suggestions. We have added information and some of the recommended citations in the introduction regarding the importance of bereavement as a risk factor in prevention of suicide, as well as in the section on “Effects of preventive interventions: summary of findings and implications”, and about biological factors as possible risk factors in the introduction, as well as in “Effects of interventions for existing self-harm: summary of findings and implications”. 2) In addition, why the authors decided to include all publications in English, Norwegian, Danish or Swedish rather than simply including only studies in English language is a matter of debate and needs to be specified. We thank reviewer 2 for pointing this out, and have added information about why choosing English, Norwegian, Danish or Swedish rather than simply including only studies in English language. For pragmatic reasons, we have included languages available to us. Furthermore, guidelines developed in Sweden, Denmark and UK carry out extensive evidence reviews. Neglecting to include these would weaken the evidence base. 3) Moreover, the authors should immediately present and discuss, in the first lines of the Discussion section, their most relevant study findings. Conversely, they seem to focus with redundancy on the main aims/objectives of the paper which have been already presented elsewhere. We agree, and are now more focused on our main findings in the beginning of the discussion. 4) Although the authors reported that the present analyses are based on secondary reporting and the interpretation of the review authors as well as that the present report included only reviews of studies where the population was children and young people with existing self-harm, the most relevant limitations/shortcomings of the present study need to be more carefully described for the general readership. We now described more carefully the most relevant limitations/shortcomings of the present study, including examples of what we mean, so that the limitations become more apparent. 5) Finally, what is the take-home message of this manuscript? While the authors stated that practice should be evaluated, and researchers should investigate harmful effects as well as beneficial effects of interventions, they failed, in my opinion, to provide some conclusive remarks about their findings. Here, some further details/information are needed. Thank you, and we agree. We now have the take-home message as well as some further details/information in conclusive remarks about the finding in the conclusion." } ] } ]	1	https://f1000research.com/articles/8-890
https://f1000research.com/articles/9-119/v1	18 Feb 20	{ "type": "Case Report", "title": "Case Report: Upper airway obstruction due to rheumatoid arthritis", "authors": [ "Mohan Rudrappa", "Laxmi Kokatnur", "Sanket Shah", "Laxmi Kokatnur", "Sanket Shah" ], "abstract": "Rheumatoid arthritis (RA) is a common autoimmune disease characterized by inflammation of small joints. Small synovial joints in the larynx can also become affected, and laryngeal involvement is seen in more than half of patients with RA. As most patients have subtle symptoms and indolent course, they are either misdiagnosed or undiagnosed. The acute worsening of cricoarytenoid arthritis can cause sudden upper airway obstruction and may require emergency intubation or tracheostomy. This life-threatening condition is described in only a handful of cases in the medical literature. Physicians should be aware of this rare but life-threatening consequence of RA. We present a case of sudden and severe upper airway obstruction secondary to laryngeal involvement in a patient with long-standing RA.", "keywords": [ "Rheumatoid arthritis", "Upper airway obstruction", "cricoarytenoid joint", "Odontoid fracture" ], "content": "Introduction\n\nRheumatoid arthritis (RA) is a common autoimmune disease that affects 1% of the adult population1. It is characterized by the development of both articular and extra-articular lesions with a predilection for small joints. Laryngeal involvement in patients with RA is invariably underdiagnosed due to its subtle clinical features, but at times it can present as a life-threatening emergency. The involvement of the cricoarytenoid joint can lead to paralysis of the vocal cord leading to upper airway obstruction. We present a case of sudden and severe upper airway obstruction secondary to laryngeal involvement in a patient with long-standing RA.\n\n\nCase presentation\n\nA 75-year-old woman with a history of coronary artery disease (status post- coronary artery bypass graft seven years ago) and RA (diagnosed ten years back being treated with 10 mg/day of prednisone and 200 mg/day of Plaquenil) was admitted to the hospital for evaluation of new-onset seizure. She was followed by a rheumatologist when she was living in Texas several years back, and presently it is managed by her primary care physician. The patient has tried several medications for her RA, including Imuran and monthly injections, and reports that only prednisone helps her arthritis.\n\nA CT scan of the head done in the Emergency Room did not show any acute intracranial process but suggested the possibility of a cervical spine fracture. Dedicated CT of the cervical spine did confirm type II odontoid fracture (Figure 1A). The patient was evaluated by Neurosurgery and opted for conservative management with a cervical collar as the patient did not have any neurological symptoms. On the night of admission to the medical floor, the patient developed hypoxemia and was started on supplemental oxygen through the nasal cannula. However, her saturation continued to deteriorate, leading to the development of severe bradycardia was transferred to the Intensive Care Unit for further management. Urgent CT pulmonary angiogram did not show any embolism nor pleuro-parenchymal changes. In the intensive care unit, the patient was initially treated with nasal cannula oxygen and later high flow oxygen without any improvement in saturation. The patient reported some breathlessness but was not in distress. Clinical examination was normal. This newly diagnosed unexplained hypoxemia was attributed to the hard cervical collar leading to airway compromise. However, despite trying a different sized hard collar and soft cervical collar, she continued to show hypoxemia. The patient was later started on with Bilevel Positive Airway Pressure (BIPAP) with improvement in saturation. By evening, the saturation dropped to 70% despite the up-titration of BiPAP settings and developed sinus bradycardia with a heart rate of 30 beats/min. The patient was emergently intubated. Due to cervical cord fracture, intubation was done with a fibro-optic pediatric bronchoscope, and seven number endotracheal tube was placed without any difficulty. Arytenoid swelling was noted during intubation, but due to emergency procedure and lack of video monitoring, it was not explored further.\n\nThe next day, video bronchoscopy performed through the endotracheal tube did not reveal any endobronchial pathology. However, bronchoscopy through the right nostril showed evidence of significant arytenoid swelling compromising the upper airway space (Figure 2). On reviewing the CT scan of the cervical spine done in the emergency room, there was evidence of arytenoid cartilage sclerosis and cricoarytenoid joint involvement secondary to RA (Figure 1B). Further collateral history was obtained from the patient's daughter, who denied any prior history of speech, swallow, or breathing problem. The patient has only multiple joint pain, stiffness, and deformity without any other organ involvement RA. The patient smoked a pack of cigarettes several years back, quit smoking seven years back when she had heart surgery. She has not been diagnosed with chronic obstructive pulmonary disease nor with any sleep-related problems before.\n\nEndotracheal tube (blue arrow) can be seen entering the vocal cords.\n\nBased on these findings, the patient was diagnosed with sudden onset of cricoarytenoid arthritis due to RA. After intubation, the patient did not show any evidence of desaturation but developed ventilator-associated pneumonia and was treated with antibiotics. The patient was treated with 60 mg/day prednisone for a week, along with antibiotics and bronchodilators. She did not show any improvement in upper airway obstruction without any air leak around the vocal cords during the air-leak test done on the ventilator. The patient underwent tracheostomy and was transferred to Long-Term Acute Care for further management after a week of hospitalization. She was later weaned from the ventilator but could not be decannulated from tracheostomy. She was discharged with 6 mm Shiley™ tracheostomy tube along with supplemental oxygen. During her follow-up visit to ENT clinic, a direct laryngoscope continued to show arytenoid swelling and the vocal cords fixed in the midline position. The patient wase also followed up with rheumatology and stared on Imuran therapy and being worked up for biological agents. Steroids were taperedd off and stopped completely.\n\n\nDiscussion\n\nLaryngeal involvement in patients with RA seen in 31–75% of patients and histological laryngeal involvement in postmortem studies can be seen in up to 90% of patients2,3. It can present as mild mucosal edema with inflammation, epiglottitis, cricoarytenoid arthritis, and rheumatoid vocal fold nodules. Cricoarytenoid joint involvement leads to significant edema and redness of the arytenoid cartilage, inter arytenoid pachydermia, impaired mobility, or fixation of the arytenoid or vocal cords4. Bilateral involvement of the cricoarytenoid joint is only seen in 13–30% of patients with RA2. Chronic inflammatory changes in the joints can result in ankylosis and decreased mobility of one or both vocal cords. Although the severity of the RA is a risk factor of laryngeal involvement, the severity of laryngeal involvement does not correlate with the disease severity5.\n\nClinical presentation depends on unilateral or bilateral involvement and the position of the vocal cords. Mild voice change, sore throat, foreign body sensation, throat swelling, aspiration, and dyspnea with stridor can be seen6. For chronic symptomatic, e.g. upper airway disease, systemic intra-articular steroids have some symptomatic benefit. Surgical management, including tracheostomy, adenoidectomy, or arytenoidopexy, may be necessary if progressive airway obstruction occurs despite medical management. Some patients may present with sudden upper airway obstruction due to acute decompensation of chronic joint inflammation superimposed by laryngeal edema due to chronically immobile vocal cords, often triggered by an infectious process5. The use of a laryngeal mask airway has been reported to exacerbate laryngeal swelling6. In mild cases, the use of local anesthesia and intra-articular corticosteroids may be beneficial. If intubation is required, bronchoscopic intubation with a small lumen endobronchial tube is recommended. The involvement of the cervical spine and RA requires careful attention in neck manipulation during intubation6.\n\nOur case was unique in several ways compared to other cases described in the literature2,3,5,6. Our patient also had atraumatic type II odontoid fracture due to RA. The prevalence of radiological cervical spine involvement in RA is seen in 20–85% of patients, and the most prevalent abnormality is atlantoaxial subluxation, followed by impaction7. Odontoid fracture is rare and only described in a few cases in the literature7–10. Prolonged steroid use can precipitate osteoporosis in the cervical spine and predispose to atraumatic fracture, as seen in our case. Our patient did not have any neurological symptoms, and hence, was treated with stabilization of the cervical spine with a hard collar. The new onset hypoxemia was initially attributed to the use of a hard collar and also posed challenges to emergency intubation. Our patient also had significant bradycardia during episodes of hypoxemia.\n\nTreatment of our case also has a limitation. Bronchoscopy was not performed after tracheostomy to look for the vocal card position. The patient developed ventilator-associated pneumonia and was requiring 50% Fio2; hence this was deferred.\n\n\nConclusion\n\nLaryngeal involvement and atlantoaxial subluxation of the cervical spine are common and well described in patients with RA. Acute cricoarytenoid arthritis leading to sudden upper airway obstruction is rare. Also, an atraumatic odontoid fracture complication due to RA very rare. Both these rare complications occurring together have not, to the best of our knowledge, been described in the medical literature. Physicians taking care of patients with RA should be aware of these rare complications, which can be life-threatening.\n\n\nConsent\n\nWritten informed consent for the publication of the article, and any associated images was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nScott DL, Wolfe F, Huizinga TW: Rheumatoid arthritis. Lancet. 2010; 376(9746): 1094–1108. PubMed Abstract \| Publisher Full Text\n\nGreco A, Fusconi M, Macri GF, et al.: Cricoarytenoid joint involvement in rheumatoid arthritis: radiologic evaluation. Am J Otolaryngol. 2012; 33(6): 753–755. PubMed Abstract \| Publisher Full Text\n\nKolman J, Morris I: Cricoarytenoid arthritis: a cause of acute upper airway obstruction in rheumatoid arthritis. Can J Anaesth. 2002; 49(7): 729–732. PubMed Abstract \| Publisher Full Text\n\nHamdan AL, El-Khatib M, Dagher W, et al.: Laryngeal involvement in rheumatoid arthritis. Middle East J Anaesthesiol. 2007; 19(2): 335–44. PubMed Abstract\n\nStojanović SP, Zivić L, Stojanović J, et al.: Total fixation of cricoarytenoid joint of a patient with rheumatoid arthritis and Hashimoto thyroiditis. Srp Arh Celok Lek. 2010; 138(3–4): 230–232. PubMed Abstract \| Publisher Full Text\n\nAntin-Ozerkis D, Evans J, Rubinowitz A, et al.: Pulmonary manifestations of rheumatoid arthritis. Clin Chest Med. 2010; 31(3): 451–478. PubMed Abstract \| Publisher Full Text\n\nLim DH, Bae SH, Choi JH, et al.: An Atraumatic Fracture of the Odontoid Process in a Newly Diagnosed Rheumatoid Arthritis Patient. J Rheum Dis. 2015; 22(2): 102–105. Publisher Full Text\n\nFindikoglu G, Ardic F, Akkaya N, et al.: A case with rheumatod arthritis and atraumatic odontoid fracture: disappearence of bony landmarks. Int J Rheum Dis. 2018; 21(11): 2041–2045. PubMed Abstract \| Publisher Full Text\n\nTakahata M, Abumi K, Sudo H, et al.: Cervical myelopathy due to atraumatic odontoid fracture in patients with rheumatoid arthritis: A case series. Mod Rheumatol. 2017; 27(5): 901–904. PubMed Abstract \| Publisher Full Text\n\nLewandrowski KU, Park PP, Baron JM, et al.: Atraumatic odontoid fractures in patients with rheumatoid arthritis. Spine J. 2006; 6(5): 529–533. PubMed Abstract \| Publisher Full Text" }	[ { "id": "68830", "date": "06 Aug 2020", "name": "Waiz Wasey", "expertise": [ "Reviewer Expertise Sleep Medicine" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article brings to light a very important issue with RA involving the upper airway. Here the description of involvement of cricoarytenoid arthritis and swelling secondary to RA is aptly done. I had written a paper indicating how RA involvement of the cervical vertebrae led to worsening of Sleep apnea. Unfortunately my patient did not have an endoscopy done to see if there was any swelling involved as described here. The obstruction here was severe enough leading to tracheostomy tube placement, which indicates a severe obstruction possibility which primary care physicians, ER and Rheumatologists should be aware of. Although the incidence is very rare, such complications do happen. The paper also brings out an interest fact that RA inflicted patients are at increased risk of Ventilator associated pneumonia secondary to their immunocomprised state from the medications.\n\nI concur that physicians taking care of patients with RA should be aware of any such complications that can arise and are potentially life threatening.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "88077", "date": "23 Aug 2021", "name": "Manish Gupta", "expertise": [ "Reviewer Expertise Otology", "Rhinology", "Laryngology", "Head Neck Disease" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case report has spelling errors in the last line of the Case Presentation section.\n\nThe case doesn't talk about the role of EMG to differentiate between vocal cord paralysis and cricoarytenoid joint fixation.\n\nTrauma to the neck should be ruled out. It may present with supraglottic edema and a fractured spine.\n\nEpisodes of seizure and possible cause are not explained.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-119
https://f1000research.com/articles/7-1831/v2	18 Feb 20	{ "type": "Research Article", "title": "Dispersion is essential in crop residue application", "authors": [ "Masato Oda" ], "abstract": "Background: Crop residue application can maintain soil fertility and sustain agriculture. However, the effects of residue application are unstable because of variable weather conditions and the residual effects of crop residue application. Furthermore, residue application often reduces crop yields. Therefore, I tried to clarify effective residue application factors in an environment which was has stable weather conditions and low residual effects. Methods: Majuro atoll, a coral sand atoll near the equator, was selected for the experiment site because of its stable weather and low residual effect of coral sand. A factorial design experiment using sweet corn was conducted based on the following four factors: fungi propagation before application, cutting residue into pieces, dispersion (or accumulation) of applied residue, and placement (on the surface or incorporation) with an equal amount of crop residue. The effects of each factors on the corn yields were evaluated using Cohen’s power analysis. Results: The dispersion showed the largest effect (1.2 in Cohen’s), which exceeded the effect of incorporation (0.7). The interaction of dispersion and incorporation showed a huge effect (4.9) on corn yield. Discussion: The effect of dispersion was not positive but it avoided the negative effects of residue clustering. Because, the toxicity of the plant residue and generation of toxic substances by anaerobic decomposition are widely known. Anaerobic decomposition occurs inside the residue clusters. However, dispersion reduced the toxicity by adsorption in soil and avoiding anaerobic decomposition. Furthermore, incorporation showed an interaction effect, but surface placement did not. Conclusion: The dispersion of crop residue enhanced the positive effect of crop residue incorporation by avoiding the toxicity from crop residue. This finding adds a new viewpoint for the controversy between conventional and conservation agriculture", "keywords": [ "Conservation agriculture", "Crop residue", "Green manure", "Marshall Islands", "Organic matter application", "Soil degradation", "Anaerobic decomposition" ], "content": "Introduction\n\nSoil degradation is a major constraint on food security (Gomiero, 2016; Lal, 2015), and the intervention points for reversing soil degradation are soil erosion and depletion of soil organic matter (Karlen & Rice, 2015). Conservation agriculture or green manure approaches are developing against this backdrop and emphasize the importance of retaining crop residue (Cherr et al., 2006; Pittelkow et al., 2015a); however, they have not yielded satisfactory results. For example, long-term trials of conservation agriculture show no particular interactions among factors such as tillage, mulch, rotation, soil texture and rainfall (Pittelkow et al., 2015b).\n\nThe main problem with crop residue application is that the decomposition of the residue can have both positive and negative effects on crop production (Kumar & Goh, 1999). Nitrogen benefits and nitrogen recovery from residues show that they have considerable potential (Kumar & Goh, 1999). However, microorganisms produce toxic substances during the decomposition of plant residue. Microorganisms metabolize these substances in aerobic soils. Nonetheless, the phytotoxic leachates of some decomposing cover crop residues have adverse effects on crops even under aerobic conditions (Bhowmik & Doll, 1982; Jessop & Stewart, 1983; Kimber, 1973a; Kimber, 1973b; Lynch, 1977; Lynch, 1978; Stirzaker & Bunn, 1996).\n\nThe application method of crop residue is a major factor in the success of the method, although the effects of organic matter application also differ by the quantity, conditions and timing (Chen et al., 2014; Tian et al., 2007). Both particle size and placement of the applied material affect the residue breakdown rate and the mineralization/immobilization process (Kumar & Goh, 1999). However, environmental factors have strong interactions with residue decomposition (Kumar & Goh, 1999). Finding effective crop residue application methods is difficult without stable weather and with the inability to control for no residual effects of organic matter, even with a focus on the application method.\n\nThe aim of this study was to clarify the most effective crop residue application method. Although, there are many factors concerning residue management (Kumar & Goh, 1999), I examined four factors (fungi propagation, cutting, dispersion and incorporation) from the viewpoint of farmers applicability. Experiments were conducted in low fertility and high microorganism’s activity environment. That enabled clear observing the effect of applied residue on crop yields.\n\n\nMethods\n\nMajuro Atoll, the capital of the Republic of the Marshall Islands, is located in the Pacific Ocean near the equator. The maximum and minimum monthly average temperatures are 29.4 to 30.2°C and 24.7 to 25.0°C, respectively. The average monthly precipitation is 169 to 356 mm, and the average annual precipitation is 3, 365 mm (CLIMATE-DATA.ORG). Coral sand, the soil of Majuro atoll, has relatively high organic matter levels in top soil due to refractory organic matter (organic carbon of 46.9 g kg−1 at 0–15 cm and 10.8 g kg−1 at 15–45 cm) (Deenik & Yost, 2006), and high percolation rates (1.4–3.5 × 10−3 m s−1) (Hunt & Peterson, 1980). Thus, the climate is stable, the soil has high permeability with no waterlogging, and frequent rainfall keeps the soil moisture close to field capacity, so applied organic matter is almost decomposed in the crop period. It is known that the residual effect of organic resources is small for sandy soils in a warm and humid climate (Chivenge et al., 2011).\n\nThe experimental field was located at Laura Farm (7°8\"34\"N, 171°2\"9\"E), which belongs to the Ministry of Resources and Development. The field size was 12.5 × 7.2 m. The plot size was 1.2 × 6.0 m, and the experimental field consisted of two sets of six plots. The government forbids the use of synthetic fertilizer and chemicals on this atoll in order to protect the underground aquifer; therefore, farmers use copra cake as fertilizer. Soil water-soluble NO3-N (0–5 cm layer; 1:2.5 soil:water extraction) was 4 µg g soil−1, as assessed by a nitrate ion meter (LAQUAtwin B-742, Horiba, Tokyo). No significant residual effect has been found in this field, although corn had been cropped three crops without fertilizer nor any chemicals from November 26, 2013 to July 28, 2014 (see raw data (Oda, 2018)).\n\nCorn residue to be used as the material for the experiment was harvested on July 28, 2014, just after the harvest of the previous crop of corn in the experimental field. The residue was divided into 12 bundles of the same fresh weights of 4.7 Mg ha−1 (2.1 Mg ha−1 in dry weight) and applied to the plots by different methods (described later). Corn (Zea mays L.) was planted in two rows at 0.5 m row spacing at a population of 3.3 plants m−2 on August 4, 2014, and harvested on October 21, 2014 (78 days after seeding). Irrigation, fertilizer and chemical applications were not performed. Hand weeding was performed 2 and 5 weeks after seeding and the weed residue was left on the soil surface of the plot.\n\nAn L12 orthogonal array design was used in the experiment (Taguchi, 1986). This design was used to ensure robust experimental results under a wide range of conditions. Only the main effects were seen in the experiments, and all the factor levels had six replications. The 12 experiments consisted of four factors (fungi propagation, cutting, dispersion and incorporation) that were randomly assigned to the experimental plots (Table 1). The four different treatments were established as follows. In the fungi propagation factor, the residue was stored in a compost house for 7 days until propagating fungi, and the remainder of the residue stored beside the field. For the cutting factor, the residue was cut into 10 cm pieces. For the dispersion factor, the residue was scattered evenly on the plots, and for the non-dispersion, the residue was placed on the center line of the plot in a swath approximately 30 cm wide. For the incorporation factor, I removed approximately 5 cm thickness of soil of the plots, then placed the residue, and covered the residue with the soil (Figure 1).\n\nThe 12 experiments consisted of four factors (fungi propagation, cutting, dispersion and incorporation), which were randomly assigned to the experimental plots.\n\nThe 12 experiments consisted of four factors (fungi propagation, cutting, dispersion and incorporation), which were randomly assigned to the experimental plots.\n\nStrong linearity must be found between returned residue quantity and the yield if the adopted factors have a robust performance (Taguchi, 1986). For validation purposes, I returned the crop residues into the same plot using the combination of the two most effective factors (dispersion and incorporation). The plot effect can confound the yields in this plot design since the quantity (0.4–2.0 Mg ha−1 in dry weight) of applied residues was proportional to the preceding yield in each plot. However, this conflict was considered to be small because the plot effect was very small in this field (see raw data (Oda, 2018)) and the residual effect of corn was small. I seeded corn on October 28, 2014 and harvested it on January 9, 2015 (73 days after seeding).\n\nI got precipitation data from the automatic weather station at the Laura Farm. I weighed the fresh weight of the whole crop and the kernels in each plot and checked the kernel/whole ratio was stable (R2 = 0.9983). The aboveground dry matter (DM) was calculated by multiplying the fresh weight to an aboveground DM/fresh-kernel-weight ratio (2.79) of an existing study (Miura & Watanabe, 2002). The aboveground residue DM was calculated by multiplying the DM/fresh ratio (0.46) of air-dried residue sample. Statistical power analysis was conducted using the effect size of Cohen’s d (Cohen, 1992) by the MS Excel 2016.\n\n\n\nd: Effect size, M: Mean, SD: Standard deviation\n\nA significance is affected by the sample size; however, Cohen's d is not affected by sample sizes and can evaluate the true effect. The p values were calculated with an unpaired, one-sided, unequal variances t-test. I evaluated the linearity of the input and the output using the coefficient of determination (R2) in a simple linear regression.\n\n\nResults\n\nThere were no irregular climate conditions during the experimental periods. The number of precipitation days for each 78-day and 73-day crop period were 58 and 54 days, respectively, and total precipitation was 1111 and 542 mm, respectively.\n\nTable 2 shows the results of the power analyses. The effect sizes of Cohen’s d < 0.2, 0.5, 0.8 and 1.2, and d > 2.0 correspond to small, medium, large, very large and huge, respectively (Cohen, 1992; Sawilowsky, 2009).\n\nThe same amount of crop residue was applied to plots by different methods (fungi propagation, cutting, dispersion and incorporation).\n\nP values: one-sided, unpaired, unequal distribution t-test (n = 6).\n\nDispersion had a larger effect (p = 0.045, Cohen’s d = 1.2) than incorporation (p = 0.223, Cohen’s d = 0.7). Fungi propagation and cutting had no effect (Cohen’s d = 0.0–0.1). I calculated the effects of dispersion and incorporation, assuming the effects of cutting and fungi propagation to be negligible. The interaction was huge (p = 0.005, Cohen’s d = 4.9), while the main effect of dispersion (Cohen’s d = 0.3) and incorporation (Cohen’s d = −0.2) were medium and small in effect size, respectively (Table 3).\n\nThe same amount of crop residue was applied to plots by different methods (fungi propagation, cutting, dispersion and incorporation). The effect sizes on corn yields were calculated neglecting the un-effective factors in Table 2. P values: one-sided, unpaired, unequal distribution t-test (n = 3).\n\nUnder the combination of dispersion and incorporation, input (the quantity of applied residue) was proportional to the output (harvested aboveground biomass) (Figure 2; R2 = 0.8963).\n\nCrop residues were returned into the same plot using the combination of the two most effective factors (dispersion and incorporation).\n\n\nDiscussion\n\nDispersion had a very large effect on corn yield. The yield was influenced by the huge effect of the interaction of dispersion and incorporation. The dispersion of crop residue application having a large effect on yield is probably a new finding.\n\nA positive effect for dispersion is surprising, given that dispersion typically increases nutrient loss. Intuitively, application of crop residues away from plants should be less effective (Stone et al., 2000). The results of this study will be reasonable if dispersion avoids the negative effect of residue clustering. This is because crop residue retained at the surface or incorporated into the soil may produce phytotoxic allelochemicals (Chung & Miller, 1995; Weston, 1996). In addition, Martin et al. (1990) reported that corn stover is itself phytotoxic. The combination of the toxicity produced by the decomposition of residue by bacteria, fungi and extract of residue results in different levels of toxicity.\n\nFor the aspect of adsorption, dispersion increases the adsorption of phytotoxic substances in soil. Adsorption is the most important soil factor controlling the fate of chemicals in the environment because it controls the chemical concentrations present in the soil solution, which is a physiological phenomenon that follows the Freundlich equation (Vidal & Bauman, 1997). This means that dispersion has a stable effect on avoiding phytotoxicity. We should pay closer attention to the dispersion of residue incorporation because tillage merely buries the residue (Staricka et al., 1991).\n\nConversely, the positive effect of incorporating crop residue on yield is because of the nutrient addition (Kumar & Goh, 1999), especially given that this experiment was conducted without fertilizer addition. Matching the amount and timing of nitrogen release with crop nitrogen demand is a key component of crop production (Robertson, 1997).\n\nThe combination of dispersion and incorporation brings a proportional yield increase to applied residue under non-fertilized conditions. In this case, the yield will reflect the nutrient applied by the residue. Furthermore, incorporation had an interactive effect, but surface placement did not. Residue incorporation, rather than surface placement, may enhance biological nitrogen fixation by reducing the inorganic N in soil (Kumar & Goh, 1999); however, further study is needed in this area.\n\n\nConclusion\n\nA stable climate with a low residual effect of soil can reveal a stable result for crop residue management. Cohen's power analysis showed the dispersion of crop residue had a very large effect on corn yield, and the effect arose from the interaction with incorporation. Dispersion will avoid phytotoxicity from anaerobic conditions inside residue clusters and clarify the positive effect of crop residue incorporation. This finding adding a new viewpoint for the controversy between conventional and conservation agriculture.\n\n\nEthics statement\n\nThis study was conducted with the permissions from the local government.\n\n\nData availability\n\nFigshare: Majuro Supplemental data, https://doi.org/10.6084/m9.figshare.6668129.v1 (Oda, 2018). Data from Experiments 3 and 4 includes data that supports the results of this article.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nI wish to thank the Ministry of Resources and Development for their cooperation, especially Mr. Jabukja Aikne and the Taiwan Technical Mission for providing seeds and tractor service. I also wish to express my deepest thanks to Mr. Susumu Yoshimura for supporting these experiments.\n\n\nReferences\n\nBhowmik PC, Doll JD: Corn and Soybean Response to Allelopathic Effects of Weed and Crop Residues1. Agron J. 1982; 74(4): 601–606. Publisher Full Text\n\nChen B, Liu E, Tian Q, et al.: Soil nitrogen dynamics and crop residues. A review. Agron Sustainable Dev. 2014; 34(2): 429–442. Publisher Full Text\n\nCherr CM, Scholberg JM, McSorley R, et al.: Green manure approaches to crop production: A synthesis. Agron J. 2006; 98(2): 302–319. Publisher Full Text\n\nChivenge P, Vanlauwe B, Six J: Does the combined application of organic and mineral nutrient sources influence maize productivity? A meta-analysis. Plant Soil. 2011; 342(1–2): 1–30. Publisher Full Text\n\nChung IM, Miller DA: Allelopathic Influence of Nine Forage Grass Extracts on Germination and Seedling Growth of Alfalfa. Agron J. 1995; 87(4): 767–772. Publisher Full Text\n\nCohen J: A power primer. Psychol Bull. 1992; 112(1): 155–159. PubMed Abstract \| Publisher Full Text\n\nDeenik JL, Yost RS: Chemical properties of atoll soils in the Marshall Islands and constraints to crop production. Geoderma. 2006; 136(3–4): 666–681. Publisher Full Text\n\nGomiero T: Soil Degradation, Land Scarcity and Food Security: Reviewing a Complex Challenge. Sustainability. 2016; 8(3): 281. Publisher Full Text\n\nHunt CD Jr, Peterson FL: WRRCTR No.126 Groundwater Resources of Kwajalein Island, Marshall Islands. 1980. Reference Source\n\nJessop RS, Stewart LW: Effects of crop residues, soil type and temperature on emergence and early growth of wheat. Plant Soil. 1983; 74(1): 101–109. Publisher Full Text\n\nKarlen DL, Rice CW: Soil Degradation: Will Humankind Ever Learn? Sustainability. 2015; 7(9): 12490–12501. Publisher Full Text\n\nKimber RWL: PHYTOTOXICITY FROM PLANT RESIDUES II . THE EFFECT OF TIME OF ROTTING OF STRAW FROM SOME GRASSES AND LEGUMES ON THE GROWTH OF WHEAT SEEDLINGS The influence of water extracts of rotted wheat straw on the growth of wheat and oat plants was described in Part. Plant Soil. 1973a; 38(2): 347–361. Reference Source\n\nKimber RWL: PHYTOTOXICITY FROM PLANT RESIDUES: III. THE RELATIVE EFFECT OF TOXINS AND NITROGEN IMMOBILIZATION ON THE GERMINATION AND GROWTH OF WHEAT. Plant Soil. 1973b; 38(3): 543–555. Publisher Full Text\n\nKumar K, Goh KM: Crop Residues and Management Practices: Effects on Soil Quality, Soil Nitrogen Dynamics, Crop Yield, and Nitrogen Recovery. Adv Agron. 1999; 68: 197–319. Publisher Full Text\n\nLal R: Restoring Soil Quality to Mitigate Soil Degradation. Sustainability. 2015; 7(5): 5875–5895. Publisher Full Text\n\nLynch JM: Phytotoxicity of acetic acid produced in the anaerobic decomposition of wheat straw. J Appl Bacteriol. 1977; 42(1): 81–87. PubMed Abstract \| Publisher Full Text\n\nLynch JM: Production and phytotoxicity of acetic acid in anaerobic soils containing plant residues. Soil Biol Biochem. 1978; 10(2): 131–135. Publisher Full Text\n\nMartin VL, McCoy EL, Dick WA: Allelopathy of Crop Residues Influences Corn Seed Germination and Early Growth. Agron J. 1990; 82(3): 555–560. Publisher Full Text\n\nMiura S, Watanabe Y: Growth and Yield of Sweet Corn with Legume Living Mulches(Agronomy). Jpn J Crop Sci. 2002; 71(1): 36–42. Publisher Full Text\n\nOda M: Majuro Supplemental data.xlsx. figshare. Dataset. 2018. http://www.doi.org/10.6084/m9.figshare.6668129.v1\n\nPittelkow CM, Liang X, Linquist BA, et al.: Productivity limits and potentials of the principles of conservation agriculture. Nature. 2015a; 517(7534): 365–368. PubMed Abstract \| Publisher Full Text\n\nPittelkow CM, Linquist BA, Lundy ME, et al.: When does no-till yield more? A global meta-analysis. Field Crops Res. 2015b; 183: 156–168. Publisher Full Text\n\nRobertson GP: Nitrogen use efficiency in row-crop agriculture: crop nitrogen use and soil nitrogen loss. Ecology in agriculture. Academic Press, New York. 1997; 347–365. Publisher Full Text\n\nSawilowsky SS: New Effect Size Rules of Thumb. J Mod Appl Stat Methods. 2009; 8(2): 597–599. Publisher Full Text\n\nStaricka JA, Allmaras RR, Nelson WW: Spatial Variation of Crop Residue Incorporated by Tillage. Soil Sci Soc Am J. 1991; 55(6): 1668–1674. Publisher Full Text\n\nStirzaker RJ, Bunn DG: Phytotoxicity of Ryegrass and Clover Cover Crops, and a Lucerne Alley Crop for No-till Vegetable Production. Biol Agric Hortic. 1996; 13(1): 83–101. Publisher Full Text\n\nStone EL, Migvar L, Robison WL: Growing Plants on Atoll Soils. 2000. Publisher Full Text\n\nTaguchi G: Introduction to quality engineering: designing quality into products and processes. 1986. Reference Source\n\nTian G, Badejo MA, Okoh AI, et al.: Effects of residue quality and climate on plant residue decomposition and nutrient release along the transect from humid forest to Sahel of West Africa. Biogeochemistry. 2007; 86(2): 217–229. Publisher Full Text\n\nVidal RA, Bauman TT: Fate of allelochemicals in the soil. Ciência Rural. 1997; 27(2): 351–357. Publisher Full Text\n\nWeston LA: Utilization of Allelopathy for Weed Management in Agroecosystems. Agron J. 1996; 88(6): 860–866. Publisher Full Text" }	[ { "id": "170344", "date": "07 Feb 2024", "name": "Valensi Kautsar", "expertise": [ "Reviewer Expertise internal nutrient cycling" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWhich groups are used to calculate Cohen’s d value? For example, to analyze the dispersion effect, the group of dispersion treatment is compared to which group for control?\n\nIn the Methods: \"all factor levels have six replicates\". Does the replication mean the number of plant replicates in one plot?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "11204", "date": "13 Apr 2024", "name": "Masato Oda", "role": "Author Response", "response": "Thank you for your precious comment. I'm sorry to be late to reply. The answer for your questions are as follows. 1. Which groups are used to calculate Cohen’s d value? For each treatment. Every treatment has 6 replication plots. The effect of each treatment is assumed as an additive effect. This study design cannot detect interactions. An interaction can be detected if the number of treatments is 2 instead of 4. So, I assumed that the study was designed for two treatments (dispersion and incorporation) because the Cohen's d for cutting or fungal propagation were very small (less than 0.1). 2. Does the replication mean the number of plant replicates in one plot? No. The replication means the number of plots. Factorial design experiment reduces the number of experimental plots by limiting the detection of interactions." }, { "c_id": "11707", "date": "19 Jun 2024", "name": "Masato Oda", "role": "Author Response", "response": "Thanks for the review. Sorry for the late response. Which groups are used to calculate Cohen’s d value? This experiment is designed using an orthogonal array, so the effect of each treatment is compared to the overall mean value. Does the replication mean the number of plant replicates in one plot? No. The number of replicates is the number of plots. The Taguchi method is a highly efficient experimental design. It is widely used in industry." } ] } ]	2	https://f1000research.com/articles/7-1831
https://f1000research.com/articles/8-1620/v1	11 Sep 19	{ "type": "Brief Report", "title": "The development of mouthwashes without anti-gonococcal activity for controlled clinical trials: an in vitro study", "authors": [ "Christophe Van Dijck", "Vicky Cuylaerts", "Piet Sollie", "Anna Spychala", "Irith De Baetselier", "Jolein Gyonne Elise Laumen", "Tania Crucitti", "Chris Kenyon", "Vicky Cuylaerts", "Piet Sollie", "Anna Spychala", "Irith De Baetselier", "Jolein Gyonne Elise Laumen", "Tania Crucitti" ], "abstract": "Background: The oropharynx plays a major role in the development and spread of antimicrobial resistant Neisseria gonorrhoeae among men who have sex with men. Trials are currently assessing the efficacy of bactericidal mouthwashes as possible therapeutic or preventive options against these pharyngeal gonococcal infections. Controlled clinical trials require the use of a placebo mouthwash without anti-gonococcal activity. So far, no such mouthwash has been described. We describe the development of a mouthwash for this purpose. Methods: The in vitro anti-gonococcal activity of Corsodyl®, Listerine Cool Mint®, Biotene®, phosphate buffered saline and six in-house placebo mouthwashes was evaluated. Three gonococcal isolates from patients with pharyngeal infection were exposed to the mouthwashes for a duration ranging from 30 seconds to 60 minutes. Isolates were then plated onto blood agar (5% horse blood) and incubated for 24 hours (5-7% CO2, 35 ± 2°C). Growth of N. gonorrhoeae was scored on a five-point scale (0 to 4). All experiments were conducted in duplicate. Results: Corsodyl® and Listerine Cool Mint® were bactericidal to all isolates. For the other mouthwashes, the median growth score after 60 minutes of exposure was 4 (interquartile range 4-4) for phosphate buffered saline; 1 (interquartile range 1-3) for Biotene®; and ranged between 0 and 2 for the in-house composed mouthwashes. An in-house composed mouthwash (Placebo 6) performed best, with a growth score of 2 (interquartile range 2-3). Conclusions: All of the evaluated potential placebo mouthwashes were bacteriostatic after gonococcal exposure of 30 to 60 minutes. In-house composed Placebo 6 showed less inhibition on gonococcal growth than Biotene® and the other in-house placebos and demonstrates, in our opinion, a good trade-off between anti-gonococcal properties and taste.", "keywords": [ "Neisseria gonorrhoeae", "gonorrhea", "pharyngitis", "gargle", "treatment", "eradication", "sexually transmitted diseases", "placebo", "randomized clinical trial", "mouthwash" ], "content": "Introduction\n\nThe importance of antimicrobial resistance (AMR) in Neisseria gonorrhoeae cannot be overstated. The bacterium is renowned for its capability to acquire AMR and has developed resistance to all classes of antimicrobials used for its treatment1. AMR frequently emerges in core groups, such as men who have sex with men (MSM)2. The pharmaecological theory of AMR states that a combination of a densely interconnected sexual network and excessive antimicrobial use is an important driver of this resistance3–5. If this theory is correct, current efforts to reduce sexually transmitted infection (STI) prevalence via expanded screening and antimicrobial therapy in MSM may paradoxically be playing an important role in the promotion of gonococcal AMR5,6.\n\nThese considerations have led to efforts to reduce the prevalence of gonococci in MSM and other core groups with non-antimicrobial products. One option is the use of an antiseptic mouthwash to decrease the oropharyngeal prevalence of gonococci (and other STIs). A modeling study showed that regular use of a mouthwash by MSM could reduce the prevalence of gonococci at different body sites7. A further consideration is that the oropharynx plays a central role in the emergence and spread of gonococcal AMR among MSM because of multiple reasons, which are reviewed elsewhere8. If a mouthwash can reduce the prevalence of oropharyngeal gonorrhoea without selecting for AMR, this may have the added benefit of reducing the probability of AMR emerging at this site4. Two randomized controlled trials (RCTs) are currently underway to assess whether regular mouth washing and gargling in MSM is able to reduce the cumulative incidence of gonorrhoea and other STIs. The OMEGA (Oral Mouthwash use to Eradicate GonorrhoeA) study is an RCT that assesses whether daily use of Listerine Zero® can reduce the incidence of pharyngeal gonorrhoea in a population of Australian MSM (ACTRN12616000247471)9. We are conducting a second RCT to assess if the use of Listerine Cool Mint® (LCM) is able to reduce the cumulative incidence of gonorrhoea (PReGo – Preventing Resistance in Gonorrhoea Study; registered at ClinicalTrials.gov with the identifier NCT03881007).\n\nThe choice of an optimal placebo is critical to the success of these RCTs. It is particularly important that a placebo is inert and has no bactericidal or bacteriostatic effect on gonococci. If it did, it would increase the probability of a false negative study outcome.\n\nSo far, no study has assessed mouthwashes for this purpose. In this paper, we describe the process of developing and testing a series of candidate placebo mouthwashes. Our aim was to find the most suitable formulation for use as a placebo in the PReGo study. The major criterion we used to assess the mouthwash was its anti-gonococcal activity.\n\n\nMethods\n\nWe used three stored isolates of Neisseria gonorrhoeae that had been previously isolated from the oropharynx of three treatment-naive women with pharyngeal infection at the STI clinic of the Institute of Tropical Medicine, Antwerp, as part of routine gonococcal surveillance monitoring. The isolates were preserved in skimmed milk and 20% glycerol at -80°C until the experiments were performed. Antimicrobial susceptibility was determined by the agar dilution method according to Clinical & Laboratory Standards Institute10.\n\nThe commercially available products Listerine Cool Mint® (LCM, containing alcohol and essential oils) and Corsodyl® (containing chlorhexidine 0.2%) were used to assess the isolate’s susceptibility to antibacterial mouthwashes.\n\nBiotene®, a commercially available mouthwash that does not contain alcohol, essential oils or chlorhexidine, was expected to have no antibacterial effect and was thus the first mouthwash to be evaluated as a potential placebo substance.\n\nSubsequently, six other potential placebo mouthwashes were manufactured by a pharmacist (Sollie Pharmacy, Antwerp) based on readily available and inexpensive ingredients that are stable at room temperature. Ingredients added to create a medicinal taste were sorbitol, sodium saccharinate, benzoic acid, ethanol, mint spiritus, raspberry extract and/or elderberry extract; ingredients added as a colorant were malachite green, raspberry extract, elderberry extract or solutio viridis. The composition of the mouthwashes is displayed in Table 1 and Table 2. Based on the properties of these ingredients, no major side effects would be expected to occur.\n\n§ 100 g Malachite green contains: 0.01 g malachite green oxalate, 99.99 g aqua conservans.\n\n$ 100 g Solutio viridis contains: 0.3 g patent blue (E131), 0.3 g tartrazine (E102), 0.15 g sodium benzoic acid, 0.1 g tartaric acid, 99.15 g purified water.\n\n* 100 g Aqua conservans contains: 0.0724 g methylparahydroxybenzoate, 0.0310 g propylparahydroxybenzoate, 0.9959 g propylene glycol, 98.901 g purified water.\n\nPhosphate buffered saline (PBS, pH 7.3 ± 0.2) was used as a negative control (inert product allowing unrestricted gonococcal growth) during every experiment.\n\nEach gonococcal isolate was brought into suspension in 3mL PBS at a 0.5 to 0.8 McFarland turbidity, corresponding to a concentration of 108 CFU/mL. From these suspensions, 100μl was then added to 900µL of each mouthwash, resulting in a concentration of 107 CFU/mL. After 30 seconds, 60 seconds, five minutes, 30 minutes and 60 minutes at ambient temperature (20 ± 5°C), 10µL aliquots were plated onto blood agar (5% horse blood) and incubated for 24 hours in a 6 ± 1% CO2 environment at 35 ± 2°C. Bacterial growth was visually scored on a semi-quantitative five-point scale, as described in Figure 1. Plating was conducted in duplicate for each isolate and all bacterial growth assessments were made by a single observer.\n\nNo statistical analysis was performed. This study did not involve any experiments on humans or animals and thus no ethical clearance was required.\n\n\nResults\n\nAll three isolates were susceptible to ceftriaxone and spectinomycin; isolates B and C had a slightly increased minimum inhibitory concentration (MIC) for azithromycin and isolate A had a high MIC for ciprofloxacin and cefixime. None of the strains produced penicillinase (Table 3).\n\nMIC, Minimum Inhibitory Concentration; determined by agar dilution method according to Clinical & Laboratory Standards Institute.\n\nAll isolates were fully susceptible to LCM and Corsodyl®; a full bactericidal effect was observed after an exposure of 30 seconds or longer (Table 4)11.\n\nNA, not assessed; IQR, interquartile range; PBS, phosphate buffered saline.\n\nExposure to Biotene® for 30 minutes or longer was found to inhibit gonococcal growth considerably (Table 4).\n\nPlacebo 1, an ethanol-containing mouthwash was designed to have a similar color and taste as LCM® but led to almost complete inhibition of gonococcal growth even after a short duration of exposure. Placebo 2 contained no ethanol and a lower amount of mint spiritus. Yet, its bacteriostatic effect was comparable to Biotene®. In order to determine if mint spiritus or malachite green were the inhibiting factors, these ingredients were sequentially omitted in Placebo 3 and 4. Raspberry extract was added to both in order to improve the taste, but this resulted in strong inhibition of gonococcal growth in both cases. Placebo 5 contained elderberry extract instead, but substantial gonococcal growth inhibition was seen here, too. Placebo 6 contained another type of colorant (solutio viridis) and showed the least bacteriostatic effect after 30 and 60 minutes of exposure (Table 4). During every experiment, there was full and confluent gonococcal growth after exposure to the negative control substance (PBS) (Table 4).\n\nWe noted a slight difference in susceptibility to the mouthwashes between the three tested gonococcal isolates. Isolate A was more susceptible to placebos 1–6 and Biotene® compared with isolates B and C. However, all strains showed equivalent susceptibility to LCM and Corsodyl® (Table 5 and Table 6). These differences were not assessed for statistical significance.\n\nIQR, interquartile range.\n\nNA, not assessed; PBS, phosphate buffered saline.\n\n\nDiscussion\n\nThe recognition of the oropharynx as a source of gonococcal transmission and the genesis of antimicrobial resistance in groups such as MSM has directed research interest towards novel non-antimicrobial methods to prevent or treat oropharyngeal gonococcal infection. Mouthwashes are one such option. In order to determine the efficacy of an intervention involving the use of a mouthwash, RCTs should be performed, and a non-bactericidal placebo is a prerequisite for these trials.\n\nCommercially available non-alcohol containing mouthwashes (like Biotene®) are an attractive option, but our experiments suggest that exposure to Biotene® for longer than five minutes may inhibit the growth of gonococci. Mouthwashes are typically used for 60 seconds but the substantivity of its ingredients may result in the antibacterial activity of mouthwashes persisting for over six hours12,13. To optimize their STI preventive potential, mouthwashes could be used pre and post sex, which could lead to multiple exposures per day. These considerations triggered the search for a placebo with minimal inhibitory effect for periods of up to 60 minutes.\n\nAll three isolates in the experiment were fully susceptible to LCM and Corsodyl® and isolate A was most susceptible to all placebo mouthwashes. Although this difference in susceptibility may have been the result of random variability, we could speculate that, in the absence of overt resistance to antiseptics, there might be a mechanism that partially protected isolate B and C from the harmful effect of some of the mouthwash constituents. Isolates B and C had a reduced susceptibility to azithromycin. This might have been due to the increased expression of an efflux mechanism such as the Mtr (multiple transferable resistance) efflux pump, which is linked to resistance to macrolides, as well as to many other substances like dyes and detergents14. We did, however, not perform genotypic assessment of the isolates used in this experiment.\n\nAfter testing multiple combinations of ingredients, we found that Placebo 6 had the least bacteriostatic effect in vitro.\n\nThe limitations of this study include the following. First, budget and time constraints did not allow to perform any in vivo evaluation of the mouthwashes. It is of particular interest to know whether participants can distinguish Placebo 6 from an antibacterial mouthwash. A formal head-to-head comparison with LCM is planned as part of the PReGo study. Second, the sample size of the current experiment was too small to statistically assess differences between isolates and between mouthwashes. We may have over- or underestimated the true effect of the mouthwashes. Additionally, the experiments were performed sequentially, which may have introduced some inter-run variation. In each experiment we did however include a PBS exposed control. Third, we did not assess bacterial growth blindly and we did not use a validated quantitative assessment method. Fourth, we used isolates from women with pharyngeal gonococcal infection, which are possibly not representative of the gonococci circulating among MSM. Their susceptibility pattern was, however, similar to that observed in most gonococcal isolates from MSM. Fifth, our in vitro findings are not necessarily representative of the in vivo setting as anatomical and biological properties may influence the effect of a mouthwash against gonococci in the throat. Bioactive molecules in saliva may, for example, have synergistic or antagonistic effects on the mouthwash’s active ingredients. Finally, we did not assess the effect of the placebo on the oropharyngeal microbiome. An increased or decreased growth of other oropharyngeal commensals might theoretically compete with gonococcal proliferation in the throat and influence gonococcal infectivity as well.\n\n\nConclusion\n\nThis experiment has shown that it is hard to develop an ideal placebo mouthwash as a range of frequently used ingredients inhibit gonococcal growth. A commercially available mouthwash like Biotene® seemed the perfect option at first but it had a bacteriostatic effect. A process of serial testing of various placebos resulted in a placebo mouthwash, which we believe demonstrates a good trade-off between anti-gonococcal properties and taste.\n\n\nData availability\n\nFigshare: In vitro gonococcal growth after exposure to mouthwashes. https://doi.org/10.6084/m9.figshare.975785911.\n\nThis project contains the following underlying data:\n\n- Data.xlsx (spreadsheet containing raw growth scores of the individual isolates after exposure to the experimental substances)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nUnemo M, Del Rio C, Shafer WM: Antimicrobial Resistance Expressed by Neisseria gonorrhoeae: A Major Global Public Health Problem in the 21st Century. Microbiol Spectr. 2016; 4(3): 213–37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLewis DA: The role of core groups in the emergence and dissemination of antimicrobial-resistant N gonorrhoeae. Sex Transm Infect. 2013; 89 Suppl 4: iv47–51. PubMed Abstract \| Publisher Full Text\n\nKenyon C, Osbak K: Certain attributes of the sexual ecosystem of high-risk MSM have resulted in an altered microbiome with an enhanced propensity to generate and transmit antibiotic resistance. Med Hypotheses. 2014; 83(2): 196–202. PubMed Abstract \| Publisher Full Text\n\nKenyon CR, Schwartz IS: Effects of Sexual Network Connectivity and Antimicrobial Drug Use on Antimicrobial Resistance in Neisseria gonorrhoeae. Emerg Infect Dis. 2018; 24(7): 1195–1203. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKenyon C: Risks of Antimicrobial Resistance in N. gonorrhoeae Associated with Intensive Screening Programs in Pre-Exposure Prophylaxis Programs. Clin Infect Dis. 2018; 67(1): 154–155. PubMed Abstract \| Publisher Full Text\n\nKenyon C: We need to consider collateral damage to resistomes when we decide how frequently to screen for chlamydia/gonorrhoea in preexposure prophylaxis cohorts. AIDS. 2019; 33(1): 155–157. PubMed Abstract \| Publisher Full Text\n\nZhang L, Regan DG, Chow EPF, et al.: Neisseria gonorrhoeae Transmission among Men Who Have Sex with Men: An Anatomical Site-Specific Mathematical Model Evaluating the Potential Preventive Impact of Mouthwash. Sex Transm Dis. 2017; 44(10): 586–592. PubMed Abstract \| Publisher Full Text\n\nLewis DA: Will targeting oropharyngeal gonorrhoea delay the further emergence of drug-resistant Neisseria gonorrhoeae strains? Sex Transm Infect. 2015; 91(4): 234–7. PubMed Abstract \| Publisher Full Text\n\nChow EPF, Walker S, Hocking JS, et al.: A multicentre double-blind randomised controlled trial evaluating the efficacy of daily use of antibacterial mouthwash against oropharyngeal gonorrhoea among men who have sex with men: the OMEGA (Oral Mouthwash use to Eradicate GonorrhoeA) study protocol. BMC Infect Dis. 2017; 17(1): 456. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCLSI: Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. 11th ed. CLSI standard M07. Wayne, PA: Clinical and Laboratory Standards Institute. 2018.\n\nVan Dijck C: In vitro gonococcal growth after exposure to mouthwashes. 2019. http://www.doi.org/10.6084/m9.figshare.9757859.v1\n\nPrada-Lopez I, Quintas V, Donos N, et al.: In situ substantivity of the essential oils in the oral cavity. Microb Pathog Strateg Combat them Sci Technol Educ (A Méndez-Vilas, Ed). 2013; 1112–1122. Reference Source\n\nTomás I, Cousido MC, García-Caballero L, et al.: Substantivity of a single chlorhexidine mouthwash on salivary flora: influence of intrinsic and extrinsic factors. J Dent. 2010; 38(7): 541–6. PubMed Abstract \| Publisher Full Text\n\nMorse SA, Lysko PG, McFarland L, et al.: Gonococcal strains from homosexual men have outer membranes with reduced permeability to hydrophobic molecules. Infect Immun. 1982; 37(2): 432–8. PubMed Abstract \| Free Full Text" }	[ { "id": "55772", "date": "11 Nov 2019", "name": "Maarten Franciscus Schim van der Loeff", "expertise": [ "Reviewer Expertise Infectious diseases", "especially sexually transmitted infections", "epidemiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis report by Van Dijck et al. seeks to evaluate the anti-gonococcal activity of several commercially available mouth washes, as well as of one commercial and several in-house produced 'placebo' mouth washes. The authors find that all tested commercially available mouth washes have some anti-gonococcal activity, and that some of the 'placebo' mouth washes did as well.\n\nThis study is important as mouth washes have been suggested as a potential tool for prevention of gonorrhoea on an individual and a population level. Trials examining the efficacy of oral mouth wash need a 'placebo' without anti-gonococcal activity. This report provides important data towards that.\nThis brief report is clearly written. The conclusions are based on the data. The limitations of the small study are clearly described in the Discussion.\nI am a physician and epidemiologist and recommend that also a microbiologist should review the manuscript.\nI have a few minor comments:\nIn the abstract it is not clear how the 5-point scale of N. gonorrhoeae growth is to be interpreted; make it explicit that 0 means no growth and 4 extensive growth.\n\nThe abstract mentions an IQR of 2-3 for placebo 6 at 60 minutes; Table 4 mentions an IQR of 1-3. Please check and correct.\n\nNot all readers may be familiar with the term \"pharmaecological\" (perhaps better spelled as \"pharma-ecological\"?) so a fuller explanation may be helpful.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5227", "date": "17 Feb 2020", "name": "Christophe Van Dijck", "role": "Author Response", "response": "We are very thankful to the reviewer for his valuable comments.Comment 1. In the abstract it is not clear how the 5-point scale of N. gonorrhoeae growth is to be interpreted; make it explicit that 0 means no growth and 4 extensive growth.Response: The abstract has been changed accordinglyComment 2. The abstract mentions an IQR of 2-3 for placebo 6 at 60 minutes; Table 4 mentions an IQR of 1-3. Please check and correct.Response: Thank you for pointing out this error. It has been corrected.Comment 3. Not all readers may be familiar with the term \"pharmaecological\" (perhaps better spelled as \"pharma-ecological\"?) so a fuller explanation may be helpful.Response: The term pharmaecological has been changed into “pharmaco-ecological” and the sentence has been rephrased to clarify the term." } ] }, { "id": "58822", "date": "04 Feb 2020", "name": "Victoria Miari", "expertise": [ "Reviewer Expertise Microbiology Laboratory Research" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall\nThis study is worthwhile as I'm aware it is difficult to design trials with adequate control solutions.\n\nThe study was written well and clearly for the most part; I have included some comments for improvement.\n\nAbstract\nPlease make clear that you are developing a \"control\" mouthwash rather than an active one.\n\nWhen you say the experiments were performed in duplicate you suggest that the experiment was performed twice. Reading on, you mention that you performed two measurements from the same experiment which is not an experimental duplicate. Can you rephrase to reflect this? You also mentioned that there is a good trade-off between anti-gonococcal activity and taste for one placebo; how did you measure this? It is not described in the main study.\n\nMethods\nYou mention PBS allows for unrestricted gonococcal growth; I do not believe this is correct; it maintains viability but not growth.\n\nInitial colony counts were not performed; this would have allowed comparison between the different mouthwashes with higher confidence. You cold have also somehow measured reduction in viability; this will need to be justified in the discussion.\n\nYou present results for penicillinase; how did you test this.\n\nResults\nCan you please put title rows on table 1?\n\nFor Table 2 it would be helpful to mention what the final volume/weight/mass the placebos were, or enter the values as percentages?\n\nTable 6 could perhaps go in supplementary files? The summary tables enough for these results.\n\nDiscussion\nWhat do you mean when you say you did not assess bacterial growth \"blindly\"? See comments in methods for what else needs to be considered in discussion.\n\nYou also mentioned experiments performed sequentially and also plating was performed in duplicate; from the results it seems like one of these was done. Can you clarify this?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5228", "date": "18 Feb 2020", "name": "Christophe Van Dijck", "role": "Author Response", "response": "We are very thankful to the reviewer for her valuable comments.Comment 1: Please make clear that you are developing a \"control\" mouthwash rather than an active one.Response: we added the term “placebo” to the abstract and the introduction to make this more clear to the reader.Comment 2: When you say the experiments were performed in duplicate you suggest that the experiment was performed twice. Reading on, you mention that you performed two measurements from the same experiment which is not an experimental duplicate. Can you rephrase to reflect this? You also mentioned that there is a good trade-off between anti-gonococcal activity and taste for one placebo; how did you measure this? It is not described in the main study.Response: indeed, only plating was conducted in duplicate. The abstract has been corrected to specify this. Taste was assessed by one of the researchers. We added a sentence to the methods’ section to reflect this.Comment 3: You mention PBS allows for unrestricted gonococcal growth; I do not believe this is correct; it maintains viability but not growth. Response: indeed, we corrected this in the manuscript.Comment 4: Initial colony counts were not performed; this would have allowed comparison between the different mouthwashes with higher confidence. You cold have also somehow measured reduction in viability; this will need to be justified in the discussion. Response: the three isolates used in this experiment were first inoculated onto GC agar. Next, the isolates were brought into 0.5 to 0.8 McFarland (10^8 CFU/mL) suspensions S1. Subsequently, 100 µL aliquots of each suspension S1 were then exposed to 900 µL of the mouthwashes/PBS, resulting in 10^7 CFU/mL suspensions S2. Then 10 µL of each suspension S2 were plated onto blood agar. Bacterial growth was assessed 24h after inoculation of these 10 µL aliquots. As this process was performed in parallel for all the mouthwashes/PBS in this experiment, we feel confident that gonococcal growth could be compared reliably between the mouthwashes. We did, however, not assess residual viability of the surviving bacterial colonies after mouthwash exposure. We added this as a limitation of the study.Comment 5: You present results for penicillinase; how did you test this.Response: a nitrocefin test was used (Nitrocefin Oxoïd, Thermo Scientific™).Comment 6: Can you please put title rows on table 1? Response: okComment 7: For Table 2 it would be helpful to mention what the final volume/weight/mass the placebos were, or enter the values as percentages? Response: okComment 8: Table 6 could perhaps go in supplementary files? The summary tables enough for these results.Response: it is F1000R policy to publish all tables within the main manuscript.Comment 9: What do you mean when you say you did not assess bacterial growth \"blindly\"? See comments in methods for what else needs to be considered in discussion.Response: The observer who assessed bacterial growth was not blinded to the ingredients of the mouthwashes. We clarified this in the discussion.Comment 10: You also mentioned experiments performed sequentially and also plating was performed in duplicate; from the results it seems like one of these was done. Can you clarify this?Response: Indeed, after testing Biotene®, we first tested Placebo 1, then Placebo 2, and so on. In each of these experiments plating of isolates A, B and C was performed in duplicate and a PBS-exposed control was included." } ] } ]	1	https://f1000research.com/articles/8-1620
https://f1000research.com/articles/8-2106/v1	16 Dec 19	{ "type": "Brief Report", "title": "DNA methylation changes in biomarker loci occur early in cancer progression", "authors": [ "Lukas Vrba", "Bernard W. Futscher", "Bernard W. Futscher" ], "abstract": "Tumor-specific DNA methylation can be used for cancer diagnostics and monitoring. We have recently reported a set of DNA methylation biomarkers that can distinguish plasma samples from lung cancer patients versus healthy controls with high sensitivity and specificity. Furthermore, the DNA methylation signal from the biomarker loci detected in plasma samples correlated with tumor size and decreased after surgical resection of lung tumors. In order to determine the timing of DNA methylation of these loci during carcinogenesis and thus the potential of the biomarkers to detect early stages of the disease we analyzed the DNA methylation of the biomarker loci in five precancerous conditions using available data from the GEO database. We found that the DNA methylation of the biomarker loci is gained early in carcinogenesis since most of the precancerous conditions already have biomarker loci hypermethylated. Moreover, these DNA methylation biomarkers are able to distinguish between precancerous lesions with malignant potential and those that stay benign where data is available. Taken together, the biomarkers have the potential to detect the earliest cancer stages; the only limitation to detection of cancer from plasma samples or other liquid biopsies is the timing when tumors start to shed enough DNA into body fluids.", "keywords": [ "DNA Methylation", "Cancer Biomarker", "Epigenetics" ], "content": "Introduction\n\nTumor cells have fundamentally different DNA methylation profile from normal cells of origin1–4. Some of these differences are tumor-specific, i.e. do not occur in any normal cell types, and thus could be used for tumor DNA identification. Since tumors shed DNA into bloodstream or other body fluids5–7, the detection of tumor-specific DNA methylation in these liquid biopsies could be utilized for non-invasive cancer diagnostics and monitoring8,9. This initiated a search for cancer-specific DNA methylation biomarker loci and analysis of these loci in plasma samples and other liquid biopsies10–12. We have previously described a large suite of cancer-specific DNA methylation biomarker loci discovered using TCGA and GEO data from over 10,000 tumor and normal samples13. Recently, we developed qPCR amplicons specific for a subset of these biomarker loci designed to detect common carcinoma types and tested them on clinical cfDNA samples from healthy individuals and non-small cell lung cancer (NSCLC) patients. We demonstrated that these biomarkers can distinguish between healthy subjects and NSCLC patients with high sensitivity and specificity14. Moreover, in blood samples from lung cancer patients the biomarker DNA methylation signal positively correlates with tumor size14. The purpose of the current study was to find how early during carcinogenesis the biomarker loci gain DNA methylation in order to assess their potential as detectors of early stage cancer. To this end, we analyzed DNA methylation of the biomarker loci in publically available data from several precancerous conditions. We found that the biomarker loci gain DNA methylation early in carcinogenesis since they are methylated already in majority precancerous lesions analyzed; in addition, where the data are available, the markers can distinguish lesions with malignant potential from those that stay benign.\n\n\nMethods\n\nThe DNA methylation data from the Illumina HumanMethylation450 platform were downloaded from the GEO database (GEO accessions GSE60185, GSE66313, GSE53051, GSE58999, GSE48684, GSE77954, GSE72872, GSE81334, GSE108123 and GSE39279). These DNA methylation data are presented as beta values - numeric values in interval 0.0-1.0. For unmethylated CpGs the beta value approaches zero, for fully methylated CpGs beta approaches 1 and for CpGs methylated in a fraction of the sample 0<beta<1, e.g. a CpG methylated in 50% of the sample will have a beta value of approximately 0.5. All data were analyzed in the R programming environment, version 3.6.115 as follows: The beta values were normalized as described13. The normalized beta values for 10 biomarker CpGs (Table 1,14) were used in further analysis. Boxplots were created using the R function boxplot and the R library beeswarm,version 0.2.3. Multidimensional scaling (MDS) plots were constructed using the R function cmdscale on matrices of distances between samples and projected into two dimensions. The ability of the marker set to distinguish between progressive and regressive lung CIS was evaluated using receiver operating characteristic (ROC) analysis on the sums of the beta values from all 10 marker CpG Illumina probes (Table 1). The ROC analysis and AUC calculations were performed using the R library pROC16, version 1.15.3.\n\nThe first column specifies Illumina CpG probe and the second column shows the genomic position of each biomarker CpG.\n\n\nResults and discussion\n\nWe have previously described a set of DNA methylation biomarker loci that are hypermethylated in 10 common carcinoma tumor types and we demonstrated that the level of DNA methylation of these loci can differentiate between plasma samples from lung cancer patients and healthy individuals. To determine the timing of the hypermethylation of these biomarker loci during human carcinogenesis and thus estimate potential of the markers to detect early disease stages we analyzed here the DNA methylation state of the biomarker loci in several premalignant conditions: breast ductal carcinoma in situ (DCIS), colorectal adenomas, Barrett’s esophagus (BE), pancreatic intraductal papillary mucinous neoplasms (IPMNs) and lung carcinoma in situ (CIS) using publically available Illumina HumanMethylation450 datasets from the GEO database.\n\nDuctal carcinoma in situ is a precursor of invasive breast carcinoma (IBC). We analyzed DNA methylation of the biomarker loci in normal breast tissue samples, DCIS and IBC from three GEO datasets: GSE6018517, GSE6631318, GSE5305119. The results (Figure 1A) show that the biomarker loci are methylated already in DCIS at about the same level as in IBC. The multidimensional scaling (MDS) plot (Figure 1B) shows DCIS samples scattered among IBC samples, indicating comparable levels of DNA methylation of individual markers, while most of the normal samples form a small cluster on a side of the plot. Furthermore, there is no significant increase in the marker methylation during the progression to metastatic disease, as illustrated by data from a cohort (GSE5899920) of 44 pairs of primary breast tumors and lymph node metastases (Figure 1A).\n\nThe figure shows data from five primary carcinoma sites: breast (A, B), colorectal (C, D), esophagus (E, F), pancreas (G, H) and lung (I, J, K). Boxplots (A, C, E, G, I) show cumulative DNA methylation of the 10 biomarker loci in normal tissue samples, precancerous lesions, and tumor samples. The y-axes of plots represent the sums of beta values of the entire set of 10 biomarker CpG Illumina probes. The breast boxplots (A) in addition show DNA methylation of the biomarker set in 44 pairs of primary breast tumors and lymph node metastases. The lung boxplots (I) in addition present the precancerous lesion (carcinoma in situ (CIS)) cohort split into progressive and regressive sub cohorts and lung tumor (squamous cell carcinoma (SCC)) cohort split into stages I-III. The multidimensional scaling (MDS) plots (B, D, F, H, K) show multidimensional scaling of pairwise distances derived from beta values of 10 biomarker CpG probes of the same sample cohorts as in the boxplots. The receiver operating characteristic (ROC) plot (J) shows that the cumulative DNA methylation of the 10 biomarkers can differentiate progressive lung CIS samples not only from normal lung tissue samples (blue curve), but also from regressive lung CIS samples (purple curve). DCIS, ductal CIS; IPMN, intraductal papillary mucinous neoplasm.\n\nColorectal adenomas are the precursor neoplasms to colorectal cancer. We analyzed biomarker loci in normal colorectal tissue, colorectal adenomas, colorectal carcinomas and metastatic colorectal tumors from three GEO datasets: GSE4868421, GSE7795422, GSE5305119. Similar to DCIS, biomarker loci are already hypermethylated in colorectal adenomas with no further increase in methylation during the progression into invasive colorectal carcinomas or metastatic colorectal cancer (Figure 1C) and again colorectal adenomas on MDS plot are scattered among colorectal carcinomas (Figure 1D).\n\nBarrett’s esophagus is a precancerous precursor of esophageal adenocarcinoma (EAC). We analyzed normal esophagus together with BE and EAC samples from two GEO datasets: GSE7287223, GSE8133424. Again, similar to DCIS or colorectal adenomas, biomarker loci are hypermethylated already in BE (Figure 1E, F). Similar situation was observed also in pancreatic intraductal papillary mucinous neoplasms (IPMNs), precursor lesions of pancreatic adenocarcinomas, where only one small dataset (GSE5305119) was available (Figure 1G, H).\n\nFinally, we analyzed lung CIS. Lung CIS is a pre-invasive precursor lesion of lung squamous cell carcinoma (SCC), one of the two non-small cell lung cancers that we previously used to demonstrate the capability of the biomarkers to distinguish between clinical plasma samples from cancer patients and healthy subjects. We analyzed DNA methylation of the biomarker loci in lung CIS together with lung SCC and normal lung tissue samples from GEO datasets GSE10812325, GSE3927926. The advantage of the original lung CIS study (GSE10812325) is that the prospective follow-up information is available for CIS samples and thus the samples could be classified as either progressive (those later progressed into invasive cancer) or regressive (these later regressed to normal epithelium or low-grade disease). Our analysis revealed, similar to the other pre-invasive lesions, that the biomarker loci have increased DNA methylation already at the lung CIS stage (Figure 1I). More importantly, when we analyzed progressive and regressive lung CIS samples separately (Figure 1I), we found that the biomarker set is able to distinguish between the two types of premalignant lesions with high sensitivity and specificity (AUC = 0.92, Figure 1J). The majority of the regressive lung CIS samples on the MDS plots cluster close to normal lung controls while all progressive lung CIS samples are scattered among lung SCC samples (Figure 1K). Even when lung SCC samples are sub-grouped into the individual cancer progression stages (I-III) there is no increase in DNA methylation with the stage (Figure 1I). Together, these results show that the gain of DNA methylation of the biomarker loci is an early epigenetic event during human carcinogenesis.\n\nThe data presented here show that DNA methylation of the biomarker loci is fundamentally changed early during the malignant progression since it is already observed in precancerous lesions. The data from lung CIS further show that the DNA methylation level of the biomarkers can differentiate between potentially malignant and benign CIS. Together, these findings indicate that the biomarkers are capable, from the qualitative point of view, to detect cancer at its earliest stages. However, the detection of cancer-specific DNA methylation in blood or other body fluids is quantitative in nature and depends on the tumor size and its propensity to shed DNA into bloodstream; e.g., our previous report14 shows that the DNA methylation signal from this biomarker set in cfDNA samples depends on the NSCLC tumor size. Later disease stages are thus relatively easy to detect since larger tumors of later cancer stages shed a large amount of DNA into bloodstream resulting in high DNA methylation signal. In order to detect the early cancer stages as well, sensitive detection techniques and especially sample processing leading to minimal background DNA methylation signal will be profound to distinguish cancer from healthy samples. This report shows that the DNA methylation change of the biomarker loci is already present to its full extent in the earliest cancer stages. Thus, the combination of the sensitive detection and the timing of the release of enough tumor DNA into blood or other body fluids are the factors that will set the limit of the biomarkers to detect cancer early.\n\nIn conclusion, the biomarker loci have the potential to detect malignant disease at its earliest stage and the only limitation to the use of the biomarkers to detect cancer from liquid biopsies is the timing when the tumors start to release enough DNA into bloodstream.\n\n\nData availability\n\nIllumina HumanMethylation450 DNA methylation data used in the presented study can be downloaded from the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/; Accession numbers: GSE60185, GSE66313, GSE53051, GSE58999, GSE48684, GSE77954, GSE72872, GSE81334, GSE108123 and GSE39279).", "appendix": "References\n\nFeinberg AP, Ohlsson R, Henikoff S: The epigenetic progenitor origin of human cancer. Nat Rev Genet. 2006; 7(1): 21–33. PubMed Abstract \| Publisher Full Text\n\nKulis M, Esteller M: DNA methylation and cancer. Adv Genet. 2010; 70: 27–56. PubMed Abstract \| Publisher Full Text\n\nSina AA, Carrascosa LG, Liang Z, et al.: Epigenetically reprogrammed methylation landscape drives the DNA self-assembly and serves as a universal cancer biomarker. Nat Commun. 2018; 9(1): 4915. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoss J, Magenheim J, Neiman D, et al.: Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease. Nat Commun. 2018; 9(1): 5068. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBettegowda C, Sausen M, Leary RJ, et al.: Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med. 2014; 6(224): 224ra24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJahr S, Hentze H, Englisch S, et al.: DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res. 2001; 61(4): 1659–65. PubMed Abstract\n\nSnyder MW, Kircher M, Hill AJ, et al.: Cell-free DNA Comprises an In Vivo Nucleosome Footprint that Informs Its Tissues-Of-Origin. Cell. 2016; 164(1–2): 57–68. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchwarzenbach H, Hoon DS, Pantel K: Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer. 2011; 11(6): 426–37. PubMed Abstract \| Publisher Full Text\n\nWan JCM, Massie C, Garcia-Corbacho J, et al.: Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer. 2017; 17(4): 223–38. PubMed Abstract \| Publisher Full Text\n\nFackler MJ, Lopez Bujanda Z, Umbricht C, et al.: Novel methylated biomarkers and a robust assay to detect circulating tumor DNA in metastatic breast cancer. Cancer Res. 2014; 74(8): 2160–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrutzmann R, Molnar B, Pilarsky C, et al.: Sensitive detection of colorectal cancer in peripheral blood by septin 9 DNA methylation assay. PLoS One. 2008; 3(11): e3759. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUehiro N, Sato F, Pu F, et al.: Circulating cell-free DNA-based epigenetic assay can detect early breast cancer. Breast Cancer Res. 2016; 18(1): 129. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVrba L, Futscher BW: A suite of DNA methylation markers that can detect most common human cancers. Epigenetics. 2018; 13(1): 61–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVrba L, Oshiro MM, Kim SS, et al.: DNA methylation biomarkers discovered in silico detect cancer in liquid biopsies from non-small cell lung cancer patients. Epigenetics. 2019; 1–12. PubMed Abstract \| Publisher Full Text\n\nTeam RC: R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing. 2019. Reference Source\n\nRobin X, Turck N, Hainard A, et al.: pROC: an open-source package for R and S+ to analyze and compare ROC curves. BMC Bioinformatics. 2011; 12: 77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFleischer T, Frigessi A, Johnson KC, et al.: Genome-wide DNA methylation profiles in progression to in situ and invasive carcinoma of the breast with impact on gene transcription and prognosis. Genome Biol. 2014; 15(8): 435. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohnson KC, Koestler DC, Fleischer T, et al.: DNA methylation in ductal carcinoma in situ related with future development of invasive breast cancer. Clin Epigenetics. 2015; 7: 75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTimp W, Bravo HC, McDonald OG, et al.: Large hypomethylated blocks as a universal defining epigenetic alteration in human solid tumors. Genome Med. 2014; 6(8): 61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReyngold M, Turcan S, Giri D, et al.: Remodeling of the methylation landscape in breast cancer metastasis. PLoS One. 2014; 9(8): e103896. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuo Y, Wong CJ, Kaz AM, et al.: Differences in DNA methylation signatures reveal multiple pathways of progression from adenoma to colorectal cancer. Gastroenterology. 2014; 147(2): 418–29.e8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQu X, Sandmann T, Frierson H Jr, et al.: Integrated genomic analysis of colorectal cancer progression reveals activation of EGFR through demethylation of the EREG promoter. Oncogene. 2016; 35(50): 6403–6415. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrause L, Nones K, Loffler KA, et al.: Identification of the CIMP-like subtype and aberrant methylation of members of the chromosomal segregation and spindle assembly pathways in esophageal adenocarcinoma. Carcinogenesis. 2016; 37(4): 356–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYu M, Maden SK, Stachler M, et al.: Subtypes of Barrett's oesophagus and oesophageal adenocarcinoma based on genome-wide methylation analysis. Gut. 2018; 68(3). pii: gutjnl-2017-314544. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTeixeira VH, Pipinikas CP, Pennycuick A, et al.: Deciphering the genomic, epigenomic, and transcriptomic landscapes of pre-invasive lung cancer lesions. Nat Med. 2019; 25(3): 517–25. PubMed Abstract \| Publisher Full Text\n\nSandoval J, Mendez-Gonzalez J, Nadal E, et al.: A prognostic DNA methylation signature for stage I non-small-cell lung cancer. J Clin Oncol. 2013; 31(32): 4140–7. PubMed Abstract \| Publisher Full Text" }	[ { "id": "57865", "date": "08 Jan 2020", "name": "Keith D. Robertson", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, Vrba and Futscher examined the DNA methylation status of candidate loci in pre-malignant stages of a variety of cancers (e.g. breast, pancreas, and esophagus). Ultimately, the authors observed that DNA hypermethylation events previously identified in full-blown cancer were already hypermethylated in the precancerous tissues. One particularly interesting outcome was that progressive lung carcinoma in situ demonstrated elevated methylation levels, while regressive CIS more closely resembled normal lung tissue, suggesting that these CpGs may actually be predictive of progression and/or play a role in carcinogenesis.\nThis is a straight-forward in silico analysis study, and seems worthy of indexing since the methylation biomarker field is moving rapidly and showing significant promise.\nThis reviewer has two concerns, however, that should be addressed:\nWhile the methylation trends are clearly qualitatively visible, statistical significance is not given for any of the comparisons.\n\nDue to pre-malignant lesions showing similar patterns as the corresponding cancer, does that reduce the efficacy of these biomarkers as the target screening population malignancy is indistinguishable from the cancer? For example, healthy individuals are not routinely screened for esophageal carcinoma, but those with Barrett's esophagus are.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5223", "date": "14 Feb 2020", "name": "Lukas Vrba", "role": "Author Response", "response": "Dear Dr. Robertson,Thank you for the prompt and generally positive review of our article. We have postponed our response until all reviews were available and a new version of the article was submitted.As to the first point raised, the other reviewer had the same suggestion and therefore we added p-values for comparisons of individual sample cohorts. All differences between normal tissues and respective premalignant lesions are highly statistically significant, while there are no significant differences in DNA methylation of the biomarker loci during further malignant progression.We especially appreciate your second point. The presence of the increased DNA methylation at marker loci may indeed raise doubts about how well the biomarkers may serve for cancer screening of individuals with premalignant conditions. We think that it depends on the type of the samples used for the screening (liquid vs tissue biopsies) and we have added the whole new paragraph discussing this topic. We thank you for raising this important point since it had to be discussed to make the study complete." } ] }, { "id": "57862", "date": "24 Jan 2020", "name": "Judd C. Rice", "expertise": [ "Reviewer Expertise Epigenetics." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this brief report by Vrba and Futscher, the authors sought to leverage their previous discovery of 10 cancer-associated DNA methylation biomarker loci to investigate the clinical potential of these biomarkers in assessing the progression of carcinogenesis. The authors analyzed several publicly available datasets that included normal tissue, precancerous lesions and tumor samples to generate in silico models of breast, colorectal, esophageal, pancreatic and lung cancer progression. The rigor of the study design is high as all datasets utilized the same technology (Illumina HumanMethylation450) with appropriate sample size of each group for analysis, except for the pancreatic samples. Although the data presented support the authors’ conclusions, at least by the eyeball test, the inclusion and description of detailed statistical analyses would increase confidence in the conclusions. The overall results indicate that aberrant methylation of the biomarker loci is an early epigenetic event of carcinogenesis, regardless of cancer type. This is an important finding that further supports the clinical potential of DNA methylation biomarkers in early cancer detection.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5224", "date": "14 Feb 2020", "name": "Lukas Vrba", "role": "Author Response", "response": "Dear Dr. Rice,Thank you for the evaluation of our study. According to your suggestion and suggestion from the other reviewer we added the p-values to Figure 1. The results now show that all the differences that made the “eyeball test” are also highly statistically significant, including in the pancreatic samples where the n is rather low but the control cohort is consistently unmethylated. We thank you for this suggestion, since the amended version of our manuscript will have higher impact among the readers." } ] } ]	1	https://f1000research.com/articles/8-2106
https://f1000research.com/articles/9-115/v1	13 Feb 20	{ "type": "Research Article", "title": "Effect of inter-aural modulation depth difference on interaural time difference thresholds for speech: An observational study", "authors": [ "Arivudainambi Pitchaimuthu", "Vibha Kanagokar", "Srividya Grama Bhagavan", "Jayashree S. Bhat", "Vibha Kanagokar", "Jayashree S. Bhat" ], "abstract": "Background: The temporal envelope (ENV) plays a vital role in conveying inter-aural time difference (ITD) in many clinical populations. However, the presence of background noise and electronic features, such as compression, reduces the modulation depth of ENV to a different degree in both ears. The effect of ENV modulation depth differences between the ears on ITD thresholds is unknown; therefore, this was the aim of the current study’s investigation. Methods: Six normally hearing young adults (age range 20-30 years) participated in the current study. Six vowel-consonant-vowel (VCV) (/aka/, /aga/, /apa/, /aba/, /ata/, /ada/) tokens were used as the probe stimuli. ENV depth of VCV tokens was smeared by 0%, 29%, and 50%, which results in 100%, 71%, and 50% of the original modulation depth. ITD thresholds were estimated as a function of the difference in temporal ENV depth between the ears, wherein in one ear the modulation depth was retained at 100% and in the other ear, the modulation depth was changed to 100%, 71%, and 50%. Results: Repeated measures of ANOVA revealed a significant main effect of interaural modulation depth differences on the ITD threshold (F(2,10)= 9.04, p= 0.006). ITD thresholds increased with an increase in the inter-aural modulation depth difference. Conclusion: Inter-aural ENV depth is critical for ITD perception.", "keywords": [ "Interaural time difference", "modulation depth", "interaural envelope difference", "lateralization", "localization", "ITD threshold", "temporal envelope" ], "content": "Introduction\n\nThe ability to locate the sound source has an important role in spatial release from masking, which results in better speech understanding in noise. When target and masker signals overlap in time but are spatially dispersed, the auditory system utilizes the spatial cues to segregate the target from the masker1. Spatial acuity in the horizontal plane is essential for speech perception in noise2. Inter-aural intensity differences (IID) and inter-aural time differences (ITD) due to the head shadow effect help determine the direction of the sound source in the horizontal plane. The coding of ITD and IID cues in the auditory system relies on the signal’s temporal envelope (ENV) and temporal fine structure (TFS). ENV refers to the amplitude fluctuations, and TFS refers to the fast frequency variations in the signal. ITD coding for low frequencies relies both on the TFS and ENV. However, at higher frequencies, TFS contributes mainly to IID coding, and ENV contributes predominantly to ITD coding3. Hence, ITD for a speech signal is effectively conveyed through ENV, as well as the TFS, in the low frequency bands. Nevertheless, in the high frequency bands, the ITD is predominantly conveyed by the ENV.\n\nITD cues are more important than IID in perceiving a signal against background noise4. In individuals with auditory disorders, such as cochlear hearing loss5 and auditory neuropathy6, the perception of TFS is affected to a greater extent than the ENV, and hence they have to rely on ITD cues present in the ENV for better localization and understanding. Even with technologically advanced hearing aids and cochlear implants, the hearing impaired still face difficulties in localization and speech understanding in noise7. Band-pass filtering and compression in hearing aids and cochlear implants reduce the ENV modulation depth and onset gradient, both of which are necessary for the perception of ITD cues8. ENV fluctuations will be reduced to a different degree in both the ears depending on the source location and compression setting. Other environmental factors, such as background noise and reverberation, also distort the modulation depth in both ears9. However, the effect of such a difference in ENV depth between the ears on ITD thresholds is unknown. Therefore, the aim of this study was to investigate the effect of inter-aural ENV depth difference on ITD thresholds for speech stimuli.\n\n\nMethods\n\nThe study was approved (Ref No: IEC KMC MLR 12-18/506) by the Institutional Ethics Committee, Kasturba Medical College, Mangalore, India. The observational study design was used to assess the ITD thresholds of participants as a function of inter-aural ENV depth difference. All the measurements were carried out in a single session. The study was conducted at the Department of Audiology & SLP, Kasturba Medical College, Mangalore, India between 19th December 2018 and 20th February 2019.\n\nSix young adults (age range 20–30 years) participated in the perceptual experiment. The sample size was determined based on the recommendation by Anderson & Vyngris for the minimum required sample size for psychophysical research10. Participants were recruited from the community through social media posts using a convenient sampling method. All participants had hearing thresholds of ≤ 15dBHL at audiometric octave frequencies. None of the participants had any history of otological and neurological disorders. Written informed consent was obtained from all participants of the study.\n\nITD threshold for speech was measured using six vowel-consonant-vowel (VCV) (/aka/, /aga/, /apa/, /aba/, /ata/, /ada/. The VCV tokens were uttered by a female speaker, and the tokens were digitally recorded using the Praat software version 6.0.2811 installed in an HP Probook 440 G3 laptop. The tokens were acquired using an omni directional microphone connected to a high fidelity external Creative Soundblaster X-fi USB sound device. The recorded tokens were subjected to ENV modifications in the MATLAB R2017a platform12 (alternatively the freely available GNU Octave, Scilab, could be used). Initially, the tokens were filtered between 80 and 7562Hz into 30 bands using third order elliptical filters. Corner frequencies of each band were determined based on the Greenwood function13. Frequency bands less than 2000 Hz were discarded to avoid the contribution of TFS cues. In the remaining bands, ENV was computed as the absolute component of Hilbert transformation14. The extracted ENV was low pass filtered at 128 Hz, and the depth of modulation was smeared by a factor of 0 %, 29%, and 50%, which results in 100%, 71%, and 50% of the original modulation depth. Finally, the output from the 30 bands was summed up.\n\nITD thresholds were estimated as a function of the difference in temporal ENV depth between the ears wherein in one ear the modulation depth was retained at 100%, and in the other ear, the modulation depth was changed to 100%, 71%, and 50% viz 100%–100%, 100%–71%, and 100%–50%. ITD thresholds were estimated separately for each of the conditions mentioned above. The stimulus presentation in one ear is delayed with reference to the stimulus presentation in the other ear. The ITD threshold is estimated as the minimum time delay required for the lateralization of the sound image. The initial presentation started with a 400μsec time delay, and the delay was adaptively varied using the transformed 2-down 1-up procedure. The time delay was increased by 25% for every negative response and decreased by 25% after two consecutive positive responses. A total of 12 reversals were administered, and midpoints of the last eight reversals were averaged to obtain the ITD threshold. Participants’ responses were acquired using a three alternative forced-choice task wherein they had to choose and indicate the sound which lateralized to the side and not the midline. For each trial, the stimulus token was randomly selected. The stimuli presentation and response acquisition was automatized using a custom-written script in MATLAB. Participants listened to the test stimuli through Sennheiser HD280 Pro headphones routed via Creative SoundBlaster X-fi USB sound device. The entire experiment was performed in a sound-treated room.\n\n\nData analysis\n\nThe ITD threshold of each participant was estimated as the geometric average of last eight reversals of the transformed up-down procedure. A repeated measure of ANOVA was used to investigate the main effect of inter-aural modulation depth differences on ITD thresholds. The level of significance for this analysis was 0.05. A series of paired ‘t’ tests were performed for post hoc pairwise comparisons. The level of significance considered for these analyses was 0.05. However the ‘p’ level was adjusted using Holm’s sequential Bonferroni procedure15 for each comparison. Statistical analyses were done using EZR version 1.35 software16.\n\n\nResult and discussion\n\nRepeated measures of ANOVA revealed a significant main effect of interaural modulation depth differences on ITD threshold (F(2,10)= 9.04, p= 0.006). Post hoc pairwise comparison performed with Holm’s sequential Bonferroni correction revealed that the ITD threshold increased when the modulation depth in one ear was reduced to 71%, and this reduction was marginally significant (t= -2.37, p= 0.06). Reducing the modulation depth from 100 to 50% further increased the ITD threshold significantly (t= - 3.24, p= 0.02). ITD threshold for 50% modulation depth was significantly different from ITD threshold for 71% modulation depth (t= -5.29, p= 0.003). These results suggest that as the difference in the interaural modulation depth increases, the ITD threshold increases. The mean and standard error of the mean for each interaural modulation depth condition is represented in Figure 1.\n\nThe current study investigated the effect of inter-aural modulation depth difference on ITD thresholds. Inter-aural modulation depth differences negatively impacted ITD thresholds. ITD thresholds increased with an increase in the inter-aural modulation depth difference. Trahiotis et al.17 indicated that the depth of amplitude modulations reaching the ears strongly influences the ITD sensitivity. The difference in the modulation depth between ears may lead to the reduction in the inter-aural ENV coherence, which would have negatively influenced the binaural processing abilities of neurons in estimating the ITD. A strong relationship between the binaural coherence and ITD cues for sound source identification has been reported in the past8,18. Results of the current study have potential implications in auditory devices such as the cochlear implant, where the ENV cues are mainly used for the perception. One limitation of the current study is the sample size. However, the small sample size could be justified as the primary aim of the current was to show the existence of an effect rather than generalizing the effect to a larger population. However, the study needs to be repeated with a large sample size to generalize the results to a large population.\n\n\nConclusion\n\nThe temporal ENV is an essential acoustic cue for conveying sound source information, which in turn helps in source segregation. The results of this study suggest that inter-aural ENV coherence in terms of ENV depth is essential for sound source perception.\n\n\nData availability\n\nHarvard Dataverse: ITD thresholds for temporal envelope smeared VCV tokens, https://doi.org/10.7910/DVN/OCLAE219.\n\nThis project contains the following underlying data:\n\n- stimuli tokens which has list of stimuli files: /aba/, /ada/, /aga/, /aka/, /apa/, /ata/.\n\n- ITD.m and ITD_data.tal contains 2 down 1 up psychophysical procedure file\n\n- resp.gui.fig and resp.gui.m files contain response acquisition file\n\n- signal.speech contains signal processing and preparation of dichotic stimuli file\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nBregman AS, McAdams S: Auditory Scene Analysis: The Perceptual Organization of Sound. J Acoust Soc Am. 1994; 95(2): 1177. Publisher Full Text\n\nWightman FL, Kistler DJ: Sound Localization. In: Yost WA, Popper AN, Fay RR, editors. Human Psychophysics. New York, NY: Springer New York; 1993; 155–92. Publisher Full Text\n\nKlein-Hennig M, Dietz M, Hohmann V, et al.: The influence of different segments of the ongoing envelope on sensitivity to interaural time delays. J Acoust Soc Am. 2011; 129(6): 3856–72. PubMed Abstract \| Publisher Full Text\n\nKerber S, Seeber BU: Localization in reverberation with cochlear implants: predicting performance from basic psychophysical measures. J Assoc Res Otolaryngol. 2013; 14(3): 379–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLorenzi C, Gilbert G, Carn H, et al.: Speech perception problems of the hearing impaired reflect inability to use temporal fine structure. Proc Natl Acad Sci U S A. 2006; 103(49): 18866–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNarne VK: Temporal processing and speech perception in noise by listeners with auditory neuropathy. Alain C, editor. PLoS One. 2013; 8(2): e55995. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWouters BJ, Doclo S, Koning R, et al.: Sound Processing for Better Coding of Monaural and Binaural Cues in Auditory Prostheses. 2013; 101(9): 1986–1997. Publisher Full Text\n\nMonaghan JJ, Krumbholz K, Seeber BU: Factors affecting the use of envelope interaural time differences in reverberation. J Acoust Soc Am. 2013; 133(4): 2288–300. PubMed Abstract \| Publisher Full Text\n\nShamma S, Lorenzi C: On the balance of envelope and temporal fine structure in the encoding of speech in the early auditory system. J Acoust Soc Am. 2013; 133(5): 2818–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnderson AJ, Vingrys AJ: Small samples: does size matter? Invest Ophthalmol Vis Sci. 2001; 42(7): 1411–3. PubMed Abstract\n\nBoersma P, Weenink D: Praat: doing phonetics by computer [Computer program]. Glot International. 2013. Reference Source\n\nMATLAB 2017a. Natick, Massachusetts: The Mathworks, Inc.; 2017. Reference Source\n\nGreenwood DD: A cochlear frequency-position function for several species--29 years later. J Acoust Soc Am. 1990; 87(6): 2592–605. PubMed Abstract \| Publisher Full Text\n\nZeng FG, Oba S, Garde S, et al.: Temporal and speech processing deficits in auditory neuropathy. Neuroreport. 1999; 10(16): 3429–35. PubMed Abstract \| Publisher Full Text\n\nHolm S: A simple sequentially rejective multiple test procedure. Scand J Stat. 1979. Reference Source\n\nKanda Y: Investigation of the freely available easy-to-use software 'EZR' for medical statistics. Bone Marrow Transplant. 2013; 48(3): 452–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrahiotis C, Bernstein LR, Akeroyd MA: Manipulating the \"straightness\" and \"curvature\" of patterns of interaural cross correlation affects listeners' sensitivity to changes in interaural delay. J Acoust Soc Am. 2001; 109(1): 321–30. PubMed Abstract \| Publisher Full Text\n\nRakerd B, Hartmann WM: Localization of sound in rooms. V. Binaural coherence and human sensitivity to interaural time differences in noise. J Acoust Soc Am. 2010; 128(5): 3052–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPitchaimuthu A, Kanagokar V, Bhagavan SG, et al.: \"ITD thresolds for temporal envelope smeared VCV tokens\". Harvard Dataverse, V2, UNF:6:0A/FfIRCJyQjc5beZwqSIQ== [fileUNF]. 2020. http://www.doi.org/10.7910/DVN/OCLAE2" }	[ { "id": "60100", "date": "19 Mar 2020", "name": "M. K. Ganapathy", "expertise": [ "Reviewer Expertise Electrophysiology tests of hearing", "pediatric audiology", "cochlear implants and bone conduction implants" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study is well structured and well executed.\nMethods are appropriately framed and signal processing is well defined. Literature indicate that ITD is drastically reduces from 1000-1500, so authors may consider filtering the signal <1500 than 2000 Hz (in case if it gives significant difference).\n\nSubject size is small, however it is discussed and justified. However, in future it can be done on larger population, so the head size differences would not lead to a large SD as seen in the current study.\n\nOverall well designed, with appropriate statistical analysis and well discussed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "66976", "date": "24 Jul 2020", "name": "James W. Dias", "expertise": [ "Reviewer Expertise Audition", "vision", "multisensory processing", "electrophysiology", "speech perception" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study investigated whether frequency modulation depth could moderate interaural temporal difference (ITD) thresholds. As modulation depth decreased from one ear to the other, interaural time-difference thresholds increased.\nThis study makes a small but meaningful contribution to the ITD literature. Though the study was conducted in a sample of normal-hearing younger adults, the results have implications for hearing replacement devices (i.e., hearing aids and cochlear implants).\nI find the methods and statistical approach to be tangible and easily understood. Though the authors address the concern in the manuscript, I am still concerned that the sample size is too small to be confident in the results. I would like to see this study replicated with a larger sample size in the future.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-115
https://f1000research.com/articles/9-111/v1	13 Feb 20	{ "type": "Opinion Article", "title": "Herbal pharmacovigilance in Nepal: challenges and recommendations", "authors": [ "Sunil Shrestha", "Krisha Danekhu", "Binaya Sapkota", "Nisha Jha", "Bhuvan KC", "Krisha Danekhu", "Binaya Sapkota", "Nisha Jha", "Bhuvan KC" ], "abstract": "Traditional herbal medicine is widely used globally. Despite its extensive use, there are no proper regulations on standardization and use of herbal medicinal products. Nepal has a rich biodiversity and demography comprising of different socio-ethnic groups. Herbal medicines are utilized prominently in Nepalese communities. These herbal products may cause side effects and adverse effects, such as nephrotoxicity, neurotoxicity with the heavy metal toxicity associated with their powdered dosage forms. The side effects of using herbal products have been documented, such as bleeding with use of Ginkgo biloba and increase in blood pressure with use of Ephedra. Regulation of herbal products is essential to promote their optimal and rational use. Standard tools are available for assessing adverse effects of herbal products from health authorities, like the World Health Organization. In Nepal, self-medication practice using traditional herbal medicines is common and includes the concomitant use of allopathic. There is no focal point to address the regulatory issues on herbal products currently in Nepal. The Department of Drug Administration in Nepal is nominated as a national pharmacovigilance center and there are no reports on adverse events from the use of herbal medicines so far. However, not having any reports does not ensure the absolute safety and effectiveness of herbal products, so vigilance is warranted. Herbal pharmacovigilance is needed for Nepal to ensure safe and effective use of herbal medicines as the current pharmacovigilance ecosystem does not capture those cases. In the Nepalese context, the absence of reporting mechanisms may have underreported adverse cases of herbal products. The present opinion article aims to discuss the use of herbal products in Nepal, the challenges associated with the adverse reaction due to herbal medicines, and recommendations to overcome these challenges", "keywords": [ "biodiversity", "herbal medicines", "herbal pharmacovigilance", "Nepal", "pharmacovigilance." ], "content": "Introduction\n\nBased on indigenous knowledge, the traditional practice of herbal medicines is widely prevalent in the world for preventive and therapeutic purposes to maintain good health and wellness of an individual1. The use of traditional medicine from natural sources is a common phenomenon in South Asia, and uses of traditional medicine, mostly of plant origin, have been widely reported among south Asian countries. The Indian system of medicines, such as the Siddha and Ayurveda, relies heavily on the products derived from plants2. Likewise, individuals in Pakistan have been using alternative therapies that are practiced by healers, clergymen, hakims, homeopaths and even quacks3\n\nHowever, none of these South Asian countries, such as Nepal, India and Pakistan, has a proper system for herbal pharmacovigilance, nor is there proper regulation and research to regulate and standardize the production and use of herbal medicinal products. Lack of stringent regulation and reporting mechanisms may have missed many reports of toxicity in the country. This article aims to discuss the use of herbal products in Nepal, the challenges associated with the adverse reaction due to herbal medicines, and recommendations to overcome these challenges.\n\n\nHerbal product use in Nepal\n\nNepal is a biodiversity-rich country because of its variation in the natural topography. Herbal medicines are widely used in Nepal, sometimes as an alternative to allopathic medicines or concomitantly with allopathic medicines. There is a general belief that traditional medicines are safe, effective and less costly compared to allopathic medicines. Nepal is also rich in culture and tradition where different ethnic groups celebrate different festivals and different medicinal plants are used on those occasions. For instance, in one of the biggest festivals, Dashain (celebrated in September-October), devotees use yellowish plant shoots called Jamara in Nepalese vernacular language (germinated from the seedlings of maize, barley, wheat, etc.)4. Wheatgrass (Tritcumaestivum linn) juice is used in Nepalese society as it contains a number of vitamins, minerals, amino acids, and vital enzymes, which play an important anticancer role5. Recently senior hepatologist in Nepal have claimed that there are severe side effects of drinking wheatgrass juice in patients6. Herbal cough syrup containing ginger, Adhatoda vasica, Glycyrrhiza glabra and Ocimum sanctum, are also commonly used in Nepal. A phase IV comparative randomized trial from Pakistan showed that cough syrup containing Adhatoda vasica has a favourable impact on cough and associated symptoms in children7.\n\nIn Nepal and elsewhere, the use of wild plants for producing herbal medicines is quite common, especially among traditional communities and tribal people. Nepal is one major supplier of wild medicinal plants in the world. Nepalese wild plants, such as Swertia chirata, Astilbe rivularis, Bergenia ciliata, Acorus calamus, Nardostachys grandiflora and Valeriana jatamansi are used by local people, traditional healers and herbal medicinal companies to prepare different types of herbal medicines8. More studies are needed to know about side effects of these herbal drug products.\n\n\nHerbal pharmacovigilance globally\n\nPharmacovigilance is the science and activities relating to the detection, assessment, understanding, and prevention of adverse effects or any other possible drug-related problem9. No medicine is free from side effects, be it allopathic or ayurvedic. Therefore, it is clear that besides therapeutic effects there may be systemic and/or topical side effects of herbal medicines in various body systems10–13. This is of great concern for health regulatory authorities, like the World Health Organization (WHO), to ensure the safe and effective use of traditional herbal medicines, which covers herbs, herbal materials, herbal preparations, and finished herbal products9,12.\n\nIn developed nations, the use of herbal medicines as a dietary supplement is gaining momentum in the neutraceuticals market, making standardization, quality control and clinical trials of herbal medicines mandatory14. However, even for the USA and European countries, setting up and maintaining a pharmacovigilance system has been a challenge because current pharmacovigilance systems are mostly focused on allopathic medicines, and herbal medicines come from countries of different origins than the country in which they are used, e.g. the USA and Europe15. Nevertheless, various tools exist to aid individuals to carry out herbal pharmacovigilance. For example, RUCMAS (Roussel Uclaf Causality Assessment Method) is a standard tool that has been widely used in many countries for assessing drug-induced and herb-induced liver injury16.\n\nThe distinction in the regulation of herbal medicines in developed and developing nations have sensitized the WHO to include traditional herbal medicines in pharmacovigilance programs for their safe and effective use, and quality control17.\n\n\nChallenges to herbal pharmacovigilance in Nepal\n\nIn Nepal, the Department of Drug Administration (DDA) has been nominated as the national pharmacovigilance center by the Uppsala Monitoring Center (UMC), Sweden, for reporting of adverse drug reactions18. Herbal (Ayurvedic) medicines in the market possess variation in quality control due to a lack quality standards and stringent regulatory environment19. The DDA raised the issue of significant heavy metal content in herbal products being marketed by street vendors in its bulletin ‘Drug Bulletin of Nepal’20; however, regulation and monitoring aspects are at a very early stage. Self-medication of traditional herbal medicines is also very common among Nepalese individuals21, and additionally, it is unknown if the concomitant use of both herbal and allopathic drugs may interact resulting in adverse drug events22,23. The public also have a general belief that these herbal medicines are safe.\n\nThough the DDA acts as a national pharmacovigilance center, there are no reports of adverse events so far due to traditional herbal medicines in Nepal. The Department of Ayurveda (DoAy) under the Ayurveda Health Policy, 1996, was developed to regulate herbal medicines in Nepal. The central Ayurvedic hospital established at Nardevi, Kathmandu in 1933, and Midwestern Regional Ayurvedic hospital established in Dang contributed to the promotion of the use of Ayurvedic herbal medicines. There are more than 216 zonal Ayurvedic dispensaries throughout the country. Herbs Production and Processing Company Ltd. was also established at Jadibudi, Kathmandu to produce herbal products. National Ayurveda Formulary has been published by DoAy24. Yet, there is no such effective implementation of policy, investigations and quality control of herbal products19,25. Stringent implementation of the policy is yet to be seen in the country.\n\nThere is a necessity for herbal pharmacovigilance in Nepal. However, several challenges exist for the development of herbal pharmacovigilance system in Nepal. Standardization of herbal products with respect to active ingredients is a tedious process that are yet to be performed in Nepal26. Reporting of adverse events of herbal medicines is not mandatory in Nepal27,28. The challenge remains the same for drugs other than Ayurvedic drugs. Underreporting of adverse drug reactions is common in Nepal as well. There are many factors that can affect the reporting of adverse drug reactions like lack of knowledge and perception among healthcare professionals, shown by a systematic review29. A study done in Nepal also suggests that there are reasons like availability of less time for these activities. Also, a lack of confidence to certify the occurrence of any adverse drug reactions has been listed as a reason for underreporting30,31.\n\nThere is an adulteration of heavy metals in herbal medicines and chances of microbial contamination, which may deteriorate the quality of herbal products32. Most herbal medicines are provided by traditional herbal practitioners in different regions of the nation, which makes it difficult to assesses severe effects of easily accessible herbal medicines in every corner of the nation. In addition, there may be problems in the identification of medicinal plants, since all the medicinal herbs present in Nepal are yet to be explored.\n\nThe study done by Poudel et al. has proposed that there is an enormous requirement for the advancement of regulations for the screening of herbal fixed dose combinations (FDCs) to ensure their safety, efficacy and optimum utilization33. Regardless of the uncertain health advantages of the herbal FDC products, these have become common practice in developing countries34.\n\n\nRecommendations to overcome challenges\n\nTo overcoming existing challenges in Nepal, we suggest the following recommendations for herbal pharmacovigilance:\n\ni. Healthcare professionals should be actively engaged in reporting adverse reactions caused by herbal medicines. Pharmacists should ask patients about herbal medicines when obtaining medication history. Physicians should be aware of patients taking any herbal products as dietary supplements or for any other purpose before prescribing any drugs and should counsel on the interaction and adverse effects of these, if any35.\n\nii. Pharmacists should communicate properly with the patient and the physician before dispensing medicines, including herbal ones. Patients should also be asked for their past drug history and any allergies using any medicine.\n\niii. Patients should be encouraged to inform their physicians or pharmacists about herbal medicines they were or are taking.\n\niv. Hospitals, both government or non-government, including Ayurvedic hospitals, should contain herbal pharmacovigilance centers. These pharmacovigilance centers should focus on reporting any possible adverse effects caused by herbal products. Healthcare professionals working in the pharmacovigilance center should be aware of drug interactions between herbal medicines and the disease of the patient.\n\nv. Education programs targeting all stakeholders, such as Ayurvedic physicians, pharmacists, target users and policymakers, should be should emphasize the use of herbal products, their therapeutic uses, side effects, interactions with conventional drugs, and any potentially fatal cases listed and this should be conducted regularly as a part of continuous medical education sessions for the prescribers.\n\nvi. Good Manufacturing Practice guidelines should be strictly implemented for herbal medicine formulation, along with a test of batch safety, inspections, and other quality regulation36.\n\nvii. National drug authorities, such as the DDA, DoAy, Nepal Ayurveda Medical Council and Nepal Health Research Council, must focus on ensuring quality control of marketed herbal products.\n\nviii. A National Pharmacovigilance Center should also emphasize the effects of traditional medicines and establish a causality relationship for the prediction of probable adverse effects due to herbal products. Social media may also play a vital role in the signal generation of suspected adverse reactions due to herbal medicines like an allopathic medications37.\n\nix. As many patients use herbal medicines as self-medication along with allopathic medications, the prescribers or pharmacists must check for drug interactions with herbal products.\n\nThe proposed implementation of herbal pharmacovigilance centers can be conducted in a similar way to already existing pharmacovigilance centers that deal with allopathic medicines. Their implementation can be conducted via the DDA in collaboration with regional Ayurvedic centers, such as Naradevi Ayurvedic Hospital, Tribhuvan University Ayurvedic Hospital, and District Ayurvedic Hospitals. We recommend that Vigiflow software can be used38, as per usual pharmacovigilance reporting practice, as this may be linked to the UMC. Pharmacists and Ayurvedic doctors can be mobilized for reporting and management of the software. The scope of the proposed herbal pharmacovigilance center can be expanded to consumer levels in households via consumer awareness programs, such as MedWatch39 in the USA. This may help address the concerns of consumers’ non-prescriptive Ayurvedic medications at the community level.\n\n\nThe way forward\n\nGlobally, herbal medicines are extensively used in health care. Regardless of whether upheld by therapeutic science or basically by many years of use, treatment with herbal medicines will stay prominent. In the quest for an alternative to allopathic medicines and with more evidence-based findings, the use of herbal medicine will increase in the future. Successful implementation of herbal pharmacovigilance is essential for developing reliable information/database on the safety of herbal medicines. It is also essential for the improvement of appropriate guidelines to ensure their safety and effective use. There is an urgent need for educational intervention on adverse reactions with regard to herbal medicine. Continuing education programs are recommended to provide the clinical practitioner's environment to redesign their insight in this quickly growing public health concern.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "References\n\nSheng-Ji P: Ethnobotanical approaches of traditional medicine studies: some experiences from Asia. Pharm Biol. 2001; 39 Suppl 1: 74–79. PubMed Abstract \| Publisher Full Text\n\nThillaivanan S, Samraj K: Challenges, constraints and opportunities in herbal medicines-a review. Int J Herb Med. 2014; 2(1): 21–24. Reference Source\n\nShaikh BT, Hatcher J: Complementary and Alternative Medicine in Pakistan: Prospects and Limitations. Evid Based Complement Alternat Med. 2005; 2(2): 139–142. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSapkota PP: Religious culture and medicinal plants: an anthropological study. Dhaulagiri Journal of Sociology and Anthropology. 2013; 7: 197–224. Publisher Full Text\n\nGore RD, Palaskar SJ, Bartake AR: Wheatgrass: Green Blood can Help to Fight Cancer. J Clin Diagn Res. 2017; 11(6): ZC40–ZC42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nhttps://www.trendsmap.com/twitter/tweet/1057664454471680000 (Accessed November 18, 2018).\n\nRehman H, Ali SA, Naveed S, et al.: An interquartile relationship between polyherbal extract based lozenges linkus a phase IV comparative randomised control trial. Pak J Pharm Sci. 2017; 30(3(Suppl)): 961–966. PubMed Abstract\n\nHasan MK, Gatto P, Jha PK: Traditional uses of wild medicinal plants and their management practices in Nepal-A study in Makawanpur district. International Journal of Medicinal and Aromatic Plants. 2013; 3(1): 102–112. Reference Source\n\nWorld Health Organization: WHO guidelines on safety monitoring of herbal medicines in pharmacovigilance systems. 2004. Reference Source\n\nCupp MJ: Herbal remedies: adverse effects and drug interactions. Am Fam Physician. 1999; 59(5): 1239–45. PubMed Abstract\n\nErnst E: Adverse effects of herbal drugs in dermatology. Br J Dermatol. 2000; 143(5): 923–929. PubMed Abstract \| Publisher Full Text\n\nGartoulla RP: Ethnomedicine and other alternative medication practices a study in medical anthropology in Nepal. 1992.\n\nKunwar RM, Shrestha KP, Bussmann RW: Traditional herbal medicine in far-west Nepal: a pharmacological appraisal. J Ethnobiol Ethnomed. 2010; 6: 35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKamboj VP: Herbal medicine. Curr Sci. 2000; 78(1): 35–39. Reference Source\n\nShaw D, Graeme L, Pierre D, et al.: Pharmacovigilance of herbal medicine. J Ethnopharmacol. 2012; 140(3): 513–518. PubMed Abstract \| Publisher Full Text\n\nDanan G, Teschke R: RUCAM in Drug and Herb Induced Liver Injury: The Update. Int J Mol Sci. 2015; 17(1): pii: E14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: The importance of pharmacovigilance. 2002. Reference Source\n\nDepartment of Drug Administration: Pharmacovigilance. Reference Source\n\nAdhikari SM, Regmi BM: Status of ayurvedic medicines available in the markets of Nepal. Nepal Health Research Council; 2009. Reference Source\n\nDepartment of Drug Administration: Drug Bulletin of Nepal. Reference Source\n\nShankar PR, Partha P, Shenoy N: Self-medication and non-doctor prescription practices in Pokhara valley, Western Nepal: a questionnaire-based study. BMC Fam Pract. 2002; 3: 17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPalaian S, Poudel A, Alam K, et al.: Initiation of social pharmacy research in Nepal: our experiences. Int J Clin Pharm. 2011; 33(4): 591–596. PubMed Abstract \| Publisher Full Text\n\nMiller LG: Herbal medicinals: selected clinical considerations focusing on known or potential drug-herb interactions. Arch Intern Med. 1998; 158(20): 2200–2211. PubMed Abstract \| Publisher Full Text\n\nNational Ayurveda Health Policy, 2052 (1996). Reference Source\n\nGewali MB, Awale S: Aspects of traditional medicine in Nepal. Japan: Institute of Natural Medicine. University of Toyama. 2008. Reference Source\n\nBarnes J: Pharmacovigilance of herbal medicines : a UK perspective. Drug Saf. 2003; 26(12): 829–851. PubMed Abstract \| Publisher Full Text\n\nJha N, Shankar PR, Bajracharya O, et al.: Adverse drug reaction reporting in a pharmacovigilance centre of Nepal. Australas Med J. 2012; 5(5): 268–71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPalaian S, Mishra P, Bista D, et al.: Safety profile of herbal drugs: urgent need for monitoring. Journal of Institute of Medicine. 2007; 28(2): 57–61. Reference Source\n\nLopez-Gongalez E, Herdeiro MT, Figuerras A: Determinants of under-reporting of adverse drug reactions: a systematic review. Drug Saf. 2009; 32(1): 19–31. PubMed Abstract \| Publisher Full Text\n\nHazell L, Shaki SA: Under-reporting of adverse drug reactions : a systematic review. Drug Saf. 2006; 29(5): 385–6. PubMed Abstract \| Publisher Full Text\n\nJha N, Rathore DS, Rathore DS, et al.: An educational intervention’s effect on healthcare professionals’ attitudes towards pharmacovigilance. Australas Med J. 2014; 7(12): 478–489. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJordan SA, Cunningham DG, Marles RJ: Assessment of herbal medicinal products: challenges, and opportunities to increase the knowledge base for safety assessment. Toxicol Appl Pharmacol. 2010; 243(2): 198–216. PubMed Abstract \| Publisher Full Text\n\nPoudel A, Alam K, Palaian S, et al.: Herbal Fixed Dose Combinations in Nepal: Growing Concerns in a Developing Country. J Clin Diagn Res. 2016; 10(10): FM01–FM03. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrisatoki R, Oguntibeju O: The role of herbal medicine use in HIV/AIDS treatment. Archives of Clinical Microbiology. 2010; 1(1). Reference Source\n\nEkor M: The growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety. Front Pharmacol. 2014; 4: 177. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: WHO guidelines on good manufacturing practices (GMP) for herbal medicines. Reference Source\n\nShrestha S, Palaian S, Shrestha B, et al.: The Potential Role of Social Media in Pharmacovigilance in Nepal: Glimpse from a Resource-limited Setting [Internet]. J Clin Diagn Res. 2019; 13(3): FE04–FE07. Publisher Full Text\n\nUppasala Monitioring Centre. VigiFlow. Reference Source\n\nUnited States Food and Drug Administration: MedWatch: The FDA Safety Information and Adverse Event Reporting Program. Reference Source" }	[ { "id": "68605", "date": "27 Aug 2020", "name": "Seyyed Ali Mozaffarpur", "expertise": [ "Reviewer Expertise Traditional Medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript can be accepted as a commentary or opinion article. Some minor revisions should be considered:\nIt was better to introduce Nepal, at first.\n\nSome of the terms appeared in the text without any explanation e.g ...ayurvedic.... Does the manuscript discuss the herbs using in Ayurveda?\n\nThe gap of knowledge that caused this opinion article should be better disclosed.\n\nSome of the sentences especially in \"Herbal product use in Nepal\" need more references\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? No\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "137633", "date": "18 May 2022", "name": "Brian Edwards", "expertise": [ "Reviewer Expertise Pharmacovigilance" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a useful summary which is easy to read with some sensible recommendations. However, there are some minor improvements in drafting which are needed as follows:\nAbstract Line 6 ‘and’ between nephrotoxicity and neurotoxicity and delete ‘the’ before ‘heavy’ Word missing after ‘allopathic’\nSection: herbal product use in Nepal ‘a’ missing before senior hepatologist\nSection Herbal pharmacovigilance globally RUCMAS should be RUCAM The public also have a general belief that these herbal medicines are safe. How do you know? What evidence for this statement?\nSection: Challenges to pharmacovigilance in Nepal 3rd paragraph ‘ is a tedious process that is yet to be’… NOT are\nRecommendations to overcome challenges Recommendation V delete ‘ be should’ before ‘emphasize’\nThe way forward Replace ‘information/database’ with ‘information and a database’\nRewrite \"Continuing education programs are recommended to provide the clinical practitioner’s environment to redesign their insight in this quickly growing public health concern.\" As \"Continuing education programs are recommended to provide the clinical practitioner with the insight into redesigning their response to this quickly growing public health concern.\"\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-111
https://f1000research.com/articles/9-109/v1	12 Feb 20	{ "type": "Method Article", "title": "Fluent genomics with plyranges and tximeta", "authors": [ "Stuart Lee", "Michael Lawrence", "Michael I. Love", "Michael Lawrence" ], "abstract": "We construct a simple workflow for fluent genomics data analysis using the R/Bioconductor ecosystem. This involves three core steps: import the data into an appropriate abstraction, model the data with respect to the biological questions of interest, and integrate the results with respect to their underlying genomic coordinates. Here we show how to implement these steps to integrate published RNA-seq and ATAC-seq experiments on macrophage cell lines. Using tximeta, we import RNA-seq transcript quantifications into an analysis-ready data structure, called the SummarizedExperiment, that contains the ranges of the reference transcripts and metadata on their provenance. Using SummarizedExperiments to represent the ATAC-seq and RNA-seq data, we model differentially accessible (DA) chromatin peaks and differentially expressed (DE) genes with existing Bioconductor packages. Using plyranges we then integrate the results to see if there is an enrichment of DA peaks near DE genes by finding overlaps and aggregating over log-fold change thresholds. The combination of these packages and their integration with the Bioconductor ecosystem provide a coherent framework for analysts to iteratively and reproducibly explore their biological data.", "keywords": [ "Gene Expression", "Chromatin Accessibility", "Workflow", "Data Integration", "Bioconductor", "plyranges", "tximeta" ], "content": "Introduction\n\nIn this workflow, we examine a subset of the RNA-seq and ATAC-seq data from Alasoo et al. (2018), a study that involved treatment of macrophage cell lines from a number of human donors with interferon gamma (IFNg), Salmonella infection, or both treatments combined. Alasoo et al. (2018) examined gene expression and chromatin accessibility in a subset of 86 successfully differentiated induced pluripotent stem cells (iPSC) lines, and compared baseline and response with respect to chromatin accessibility and gene expression at specific quantitative trait loci (QTL). The authors found that many of the stimulus-specific expression QTL were already detectable as chromatin QTL in naive cells, and further hypothesize about the nature and role of transcription factors implicated in the response to stimulus.\n\nWe will perform a much simpler analysis than the one found in Alasoo et al. (2018), using their publicly available RNA-seq and ATAC-seq data (ignoring the genotypes). We will examine the effect of IFNg stimulation on gene expression and chromatin accessibility, and look to see if there is an enrichment of differentially accessible (DA) ATAC-seq peaks in the vicinity of differentially expressed (DE) genes. This is plausible, as the transcriptomic response to IFNg stimulation may be mediated through binding of regulatory proteins to accessible regions, and this binding may increase the accessibility of those regions such that it can be detected by ATAC-seq.\n\nThroughout the workflow (Figure 1), we will use existing Bioconductor infrastructure to understand these datasets. In particular, we will emphasize the use of the Bioconductor packages plyranges and tximeta. The plyranges package fluently transforms data tied to genomic ranges using operations like shifting, window construction, overlap detection, etc. It is described by Lee et al. (2019) and leverages underlying core Bioconductor infrastructure (Lawrence et al., 2013; Huber et al., 2015) and the tidyverse design principles Wickham et al. (2019).\n\nFirst, we import data as a SummarizedExperiment object, which enables interoperability with downstream analysis packages. Then we model our assay data, using the existing Bioconductor packages DESeq2 and limma. We take the results of our models for each assay with respect to their genomic coordinates, and integrate them. First, we compute the overlap between the results of each assay, then aggregate over the combined genomic regions, and finally summarize to compare enrichment for differentially expressed genes to non differentially expressed genes. The final output can be used for downstream visualization or further transformation.\n\nThe tximeta package described by Love et al. (2019) is used to read RNA-seq quantification data into R/Bioconductor, such that the transcript ranges and their provenance are automatically attached to the object containing expression values and differential expression results.\n\nThe data used in this workflow is available from two packages: the macrophage Bioconductor ExperimentData package and from the workflow package fluentGenomics (Lee & Love, 2020).\n\nThe macrophage package contains RNA-seq quantification from 24 RNA-seq samples, a subset of the RNA-seq samples generated and analyzed by Alasoo et al. (2018). The paired-end reads were quantified using Salmon (Patro et al., 2017), using the Gencode 29 human reference transcripts (Frankish et al., 2019). For more details on quantification, and the exact code used, consult the vignette of the macrophage package. The package also contains the Snakemake file that was used to distribute the Salmon quantification jobs on a cluster (Köster & Rahmann, 2012).\n\nThe fluentGenomics package (Lee & Love, 2020) contains functionality to download and generate a cached SummarizedExperiment object from the normalized ATAC-seq data provided by Alasoo & Gaffney (2017). This object contains all 145 ATAC-seq samples across all experimental conditions as analyzed by Alasoo et al. (2018). The data can be also be downloaded directly from the Zenodo deposition.\n\nThe following code loads the path to the cached data file, or if it is not present, will create the cache and generate a SummarizedExperiment using the the BiocFileCache package (Shepherd & Morgan, 2019).\n\n\n\nWe can then read the cached file and assign it to an object called atac.\n\n\n\nA precise description of how we obtained this SummarizedExperiment object can be found in Importing ATAC-seq data as a SummarizedExperiment object.\n\n\nImport data as a SummarizedExperiment\n\nFirst, we specify a directory dir, where the quantification files are stored. You could simply specify this directory with:\n\n\n\nwhere the path is relative to your current R session. However, in this case we have distributed the files in the macrophage package. The relevant directory and associated files can be located using system.file.\n\n\n\nInformation about the experiment is contained in the coldata.csv file. We leverage the dplyr and readr packages (as part of the tidyverse) to read this file into R (Wickham et al., 2019). We will see later that plyranges extends these packages to accommodate genomic ranges.\n\n\n\n\n\nAfter we have read the coldata.csv file, we select relevant columns from this table, create a new column called files, and transform the existing line and condition columns into factors. In the case of condition, we specify the “naive” cell line as the reference level. The files column points to the quantifications for each observation – these files have been gzipped, but would typically not have the ‘gz’ ending if used from Salmon directly. One other thing to note is the use of the pipe operator,%>%, which can be read as “then”, i.e. first read the data, then select columns, then mutate them.\n\nNow we have a table summarizing the experimental design and the locations of the quantifications. The following lines of code do a lot of work for the analyst: importing the RNA-seq quantification (dropping inferential replicates in this case), locating the relevant reference transcriptome, attaching the transcript ranges to the data, and fetching genome information. Inferential replicates are especially useful for performing transcript-level analysis, but here we will use a point estimate for the per-gene counts and perform gene-level analysis.\n\nThe result is a SummarizedExperiment object.\n\n\n\nOn a machine with a working internet connection, the above command works without any extra steps, as the tximeta function obtains any necessary metadata via FTP, unless it is already cached locally. The tximeta package can also be used without an internet connection, in this case the linked transcriptome can be created directly from a Salmon index and gtf.\n\n\n\nBecause tximeta knows the correct reference transcriptome, we can ask tximeta to summarize the transcript-level data to the gene level using the methods of Soneson et al. (2015).\n\n\n\nOne final note is that the start of positive strand genes and the end of negative strand genes is now dictated by the genomic extent of the isoforms of the gene (so the start and end of the reduced GRanges). Another alternative would be to either operate on transcript abundance, and perform differential analysis on transcript (and so avoid defining the TSS of a set of isoforms), or to use gene-level summarized expression but to pick the most representative TSS based on isoform expression.\n\nThe SummarizedExperiment object containing ATAC-seq peaks can be created from the following tab-delimited files from Alasoo & Gaffney (2017):\n\nThe sample metadata: ATAC_sample_metadata.txt.gz (<1M)\n\nThe matrix of normalized read counts: ATAC_cqn_matrix.txt.gz (109M)\n\nThe annotated peaks: ATAC_peak_metadata.txt.gz (5.6M)\n\nTo begin, we read in the sample metadata, following similar steps to those we used to generate the coldata table for the RNA-seq experiment:\n\n\n\nThe ATAC-seq counts have already been normalized with cqn (Hansen et al., 2012) and log2 transformed. Loading the cqn-normalized matrix of log2 transformed read counts takes ~30 seconds and loads an object of ~370 Mb. We set the column names so that the first column contains the rownames of the matrix, and the remaining columns are the sample identities from the atac_coldata object.\n\n\n\nWe read in the peak metadata (locations in the genome), and convert it to a GRanges object. The as_granges() function automatically converts the data.frame into a GRanges object. From that result, we extract the peak_id column and set the genome information to the build “GRCh38”. We know this from the Zenodo entry.\n\n\n\nFinally, we construct a SummarizedExperiment object. We place the matrix into the assays slot as a named list, the annotated peaks into the row-wise ranges slot, and the sample metadata into the column-wise data slot:\n\n\n\n\nModel assays\n\nWe can easily run a differential expression analysis with DESeq2 using the following code chunks (Love et al., 2014). The design formula indicates that we want to control for the donor baselines (line) and test for differences in gene expression on the condition. For a more comprehensive discussion of DE workflows in Bioconductor see Love et al. (2016) and Law et al. (2018).\n\n\n\nThe model is fit with the following line of code:\n\n\n\nBelow we set the contrast on the condition variable, indicating we are estimating the log2 fold change (LFC) of IFNg stimulated cell lines against naive cell lines. We are interested in LFCs greater than 1 at a nominal false discovery rate (FDR) of 1%.\n\n\n\nTo see the results of the expression analysis, we can generate a summary table and an MA plot (Figure 2):\n\n\n\nGenes that have an adjusted p-value below 0.01 are colored red.\n\nWe now output the results as a GRanges object, and due to the conventions of plyranges, we construct a new column called gene_id from the row names of the results. Each row now contains the genomic region (seqnames, start, end, strand) along with corresponding metadata columns (the gene_id and the results of the test). Note that tximeta has correctly identified the reference genome as “hg38”, and this has also been added to the GRanges along the results columns. This kind of book-keeping is vital once overlap operations are performed to ensure that plyranges is not comparing across incompatible genomes.\n\n\n\nFrom this, we can restrict the results to those that meet our FDR threshold and select (and rename) the metadata columns we’re interested in:\n\n\n\nWe now wish to extract genes for which there is evidence that the LFC is not large. We perform this test by specifying an LFC threshold and an alternative hypothesis (altHypothesis) that the LFC is less than the threshold in absolute value. To visualize the result of this test, you can run results without format=\"GRanges\", and pass this object to plotMA as before.\n\nWe label these genes as other_genes and later as “non-DE genes”, for comparison with our de_genes set.\n\n\n\nThe following section describes the process we have used for generating a GRanges object of differential peaks from the ATAC-seq data in Alasoo et al. (2018).\n\nThe code chunks for the remainder of this section are not run.\n\nFor assessing differential accessibility, we run limma (Smyth, 2004), and generate the a summary of LFCs and adjusted p-values for the peaks:\n\n\n\nWe now take the rowRanges of the SummarizedExperiment and attach the LFCs and adjusted p-values from limma, so that we can consider the overlap with differential expression. Note that we set the genome build to “hg38” and restyle the chromosome information to use the “UCSC” style (e.g. “chr1”, “chr2”, etc.). Again, we know the genome build from the Zenodo entry for the ATAC-seq data.\n\n\n\nThe final GRanges object containing the DA peaks is included in the workflow package and can be loaded as follows:\n\n\n\n\nIntegrate ranges\n\nWe have already used plyranges a number of times above, to filter, mutate, and select on GRanges objects, as well as ensuring the correct genome annotation and style has been used. The plyranges package provides a grammar for performing transformations of genomic data (Lee et al., 2019). Computations resulting from compositions of plyranges “verbs” are performed using underlying, highly optimized range operations in the GenomicRanges package (Lawrence et al. 2013).\n\nFor the overlap analysis, we filter the annotated peaks to have a nominal FDR bound of 1%.\n\n\n\nWe now have GRanges objects that contain DE genes, genes without strong signal of DE, and DA peaks. We are ready to answer the question: is there an enrichment of DA ATAC-seq peaks in the vicinity of DE genes compared to genes without sufficient DE signal?\n\nAs plyranges is built on top of dplyr, it implements methods for many of its verbs for GRanges objects. Here we can use slice to randomly sample the rows of the other_genes. The sample.int function will generate random samples of size equal to the number of DE-genes from the number of rows in other_genes:\n\n\n\nWe can repeat this many times to create many samples via replicate. By replicating the sub-sampling multiple times, we minimize the variance on the enrichment statistics induced by the sampling process.\n\n\n\nThis creates a list of GRanges objects as a list, and we can bind these together using the bind_ranges function. This function creates a new column called “resample” on the result that identifies each of the input GRanges objects:\n\n\n\nSimilarly, we can then combine the boot_genes GRanges, with the DE GRanges object. As the resample column was not present on the DE GRanges object, this is given a missing value which we recode to a 0 using mutate()\n\n\n\nNow we would like to modify our gene ranges so they contain the 10 kilobases on either side of their transcription start site (TSS). There are many ways one could do this, but we prefer an approach via the anchoring methods in plyranges. Because there is a mutual dependence between the start, end, width, and strand of a GRanges object, we define anchors to fix one of start and end, while modifying the width. As an example, to extract just the TSS, we can anchor by the 5’ end of the range and modify the width of the range to equal 1.\n\n\n\nAnchoring by the 5’ end of a range will fix the end of negatively stranded ranges, and fix the start of positively stranded ranges.\n\nWe can then repeat the same pattern but this time using anchor_center() to tell plyranges that we are making the TSS the midpoint of a range that has total width of 20 kb, or 10 kb both upstream and downstream of the TSS.\n\n\n\nWe are now ready to compute overlaps between RNA-seq genes (our DE set and bootstrap sets) and the ATAC-seq peaks. In plyranges, overlaps are defined as joins between two GRanges objects: a left and a right GRanges object. In an overlap join, a match is any range on the left GRanges that is overlapped by the right GRanges. One powerful aspect of the overlap joins is that the result maintains all (metadata) columns from each of the left and right ranges which makes downstream summaries easy to compute.\n\nTo combine the DE genes with the DA peaks, we perform a left overlap join. This returns to us the all_genes ranges (potentially with duplication), but with the metadata columns from those overlapping DA peaks. For any gene that has no overlaps, the DA peak columns will have NA’s.\n\n\n\nNow we can ask, how many DA peaks are near DE genes relative to “other” non-DE genes? A gene may appear more than once in genes_olap_peaks, because multiple peaks may overlap a single gene, or because we have re-sampled the same gene more than once, or a combination of these two cases.\n\nFor each gene (that is the combination of chromosome, the start, end, and strand), and the “origin” (DE vs not-DE) we can compute the distinct number of peaks for each gene and the maximum peak based on LFC. This is achieved via reduce_ranges_directed, which allows an aggregation to result in a GRanges object via merging neighboring genomic regions. The use of the directed suffix indicates we’re maintaining strand information. In this case, we are simply merging ranges (genes) via the groups we mentioned above. We also have to account for the number of resamples we have performed when counting if there are any peaks, to ensure we do not double count the same peak:\n\n\n\nWe can then filter genes if they have any peaks and compare the peak fold changes between non-DE and DE genes using a boxplot (Figure 3):\n\n\n\nIn general, the DE genes have larger maximum DA fold changes relative to the non-DE genes.\n\nNext we examine how thresholds on the DA LFC modify the enrichment we observe of DA peaks near DE or non-DE genes. First, we want to know how the number of peaks within DE genes and non-DE genes change as we change threshold values on the peak LFC. As an example, we could compute this by arbitrarily chosen LFC thresholds of 1 or 2 as follows:\n\n\n\nHere we see that DE genes tend to have more DA peaks near them, and that the number of DA peaks decreases as we increase the DA LFC threshold (as expected). We now show how to compute the ratio of peak counts from DE compared to non-DE genes, so we can see how this ratio changes for various DA LFC thresholds.\n\nFor all variables except for the origin column we divide the first row’s values by the second row, which will be the enrichment of peaks in DE genes compared to other genes. This requires us to reshape the summary table from long form back to wide form using the tidyr package. First, we pivot the results of the peak_count columns into name-value pairs, then pivot again to place values into the origin column. Then we create a new column with the relative enrichment:\n\n\n\nThe above table shows that relative enrichment increases for a larger LFC threshold.\n\nDue to the one-to-many mappings of genes to peaks, it is unknown if we have the same number of DE genes participating or less, as we increase the threshold on the DA LFC. We can examine the number of genes with overlapping DA peaks at various thresholds by grouping and aggregating twice. First, the number of peaks that meet the thresholds are computed within each gene, origin, and resample group. Second, within the origin column, we compute the total number of peaks that meet the DA LFC threshold and the number of genes that have more than zero peaks (again averaging over the number of resamples).\n\n\n\nTo do this for many thresholds is cumbersome and would create a lot of duplicate code. Instead we create a single function called count_above_threshold that accepts a variable and a vector of thresholds, and computes the sum of the absolute value of the variable for each element in the thresholds vector.\n\n\n\nThe above function will compute the counts for any arbitrary threshold, so we can apply it over possible LFC thresholds of interest. We choose a grid of one hundred thresholds based on the range of absolute LFC values in the da_peaks GRanges\n\nobject:\n\n\n\nThe peak counts for each threshold are computed as a new list-column called value. First, the GRanges object has been grouped by the gene, origin, and the number of resamples columns. Then we aggregate over those columns, so each row will contain the peak counts for all of the thresholds for a gene, origin, and resample. We also maintain another list-column that contains the threshold values.\n\n\n\nNow we can expand these list-columns into a long GRanges object using the expand_ranges() function. This function will unlist the value and threshold columns and lengthen the resulting GRanges object. To compute the peak and gene counts for each threshold, we apply the same summarization as before:\n\n\n\nAgain we can compute the relative enrichment in LFCs in the same manner as before, by pivoting the results to long form then back to wide form to compute the enrichment. We visualize the peak enrichment changes of DE genes relative to other genes as a line chart (Figure 4):\n\n\n\nWe computed the sum of DA peaks near the DE genes, for increasing LFC thresholds on the accessibility change. As we increased the threshold, the number of total peaks went down (likewise the mean number of DA peaks per gene). It is also likely the number of DE genes with a DA peak nearby with such a large change went down. We can investigate this with a plot (Figure 5) that summarizes many of the aspects underlying the enrichment plot above.\n\n\n\nLines are colored according to whether they represent a gene that is DE or not. Note the x-axis is on a log10 scale.\n\n\nDiscussion\n\nWe have shown that by using plyranges and tximeta (with support of Bioconductor and tidyverse ecosystems) we can fluently iterate through the biological data science workflow: from import, through to modeling, and data integration.\n\nThere are several further steps that would be interesting to perform in this analysis; for example, we could modify window size around the TSS to see how it affects enrichment, and vary the FDR cut-offs for both the DE gene and DA peak sets. We could also have computed variance in addition to the mean of the bootstrap set, and so drawn an interval around the enrichment line.\n\nFinally, our workflow illustrates the benefits of using appropriate data abstractions provided by Bioconductor such as the SummarizedExperiment and GRanges. These abstractions provide users with a mental model of their experimental data and are the building blocks for constructing the modular and iterative analyses we have shown here. Consequently, we have been able to interoperate many decoupled R packages (from both Bioconductor and the tidyverse) to construct a seamless end-to-end workflow that is far too specialized for a single monolithic tool.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nSoftware availability\n\nplyranges is available from Bioconductor: https://doi.org/doi:10.18129/B9.bioc.plyranges.\n\ntximeta is available from Bioconductor: https://doi.org/doi:10.18129/B9.bioc.tximeta.\n\nSource code and all workflow materials are available at: https://github.com/sa-lee/fluentGenomics.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.3633505 (Lee & Love, 2020).\n\nLicense: MIT License.\n\nThe development version of the workflow and all downstream dependencies can be installed using the BiocManager package by running:\n\n\n\nThis article and the analyses were performed with R (R Core Team, 2019) using the rmarkdown (Allaire et al., 2019), and knitr (Xie, 2019; Xie, 2015) packages.\n\n", "appendix": "Author contributions\n\n\n\nAll authors contributed to the writing and development of the workflow.\n\n\nAcknowledgements\n\nThe authors would like to thank all participants of the Bioconductor 2019 and BiocAsia 2019 conferences who attended and provided feedback on early versions of this workflow paper.\n\n\nReferences\n\nAlasoo K, Rodrigues J, Mukhopadhyay S, et al.: Shared genetic effects on chromatin and gene expression indicate a role for enhancer priming in immune response. Nat Genet. 2018; 50(3): 424–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlasoo K, Gaffney D: Processed read counts from macrophage RNA-seq and ATAC-seq experiments. Zenodo. 2017. Publisher Full Text\n\nAllaire JJ, Xie Y, McPherson J, et al.: Rmarkdown: Dynamic Documents for R. 2019. Reference Source\n\nFrankish A, Diekhans M, Ferreira AM, et al.: GENCODE reference annotation for the human and mouse genomes. Nucleic Acids Res. 2019; 47(D1): D766–D773. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHansen KD, Irizarry RA, Wu Z: Removing technical variability in RNA-seq data using conditional quantile normalization. Biostatistics. 2012; 13(2): 204–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuber W, Carey VJ, Gentleman R, et al.: Orchestrating high-throughput genomic analysis with Bioconductor. Nat Methods. Springer Nature. 2015; 12(2): 115–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKöster J, Rahmann S: Snakemake--a scalable bioinformatics workflow engine. Bioinformatics. 2012; 28(19): 2520–2. PubMed Abstract \| Publisher Full Text\n\nLaw CW, Alhamdoosh M, Su S, et al.: RNA-seq analysis is easy as 1-2-3 with limma, Glimma and edgeR [version 3; peer review: 3 approved]. F1000 Res. F1000 Research Limited. 2018; 5: 1408. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLawrence M, Huber W, Pagès H, et al.: Software for computing and annotating genomic ranges. PLoS Comput Biol. 2013; 9(8): e1003118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLove M, Mike L: sa-lee/fluentGenomics: Bioconductor @ accepted version. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3633505\n\nLee S, Cook D, Lawrence M: plyranges: a grammar of genomic data transformation. Genome Biol. 2019; 20(1): 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLove MI, Anders S, Kim V, et al.: RNA-Seq workflow: gene-level exploratory analysis and differential expression [version 1; peer review: 2 approved]. F1000 Res. F1000 Research Limited. 2016; 4: 1070. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLove MI, Soneson C, Hickey PF, et al.: Tximeta: Reference Sequence Checksums for Provenance Identification in RNA-seq. bioRxiv. 2019; 777888. Publisher Full Text\n\nLove MI, Huber W, Anders S: Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15(12): 550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatro R, Duggal G, Love MI, et al.: Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017; 14(4): 417–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nR Core Team: R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing. 2019. Reference Source\n\nShepherd L, Morgan M: BiocFileCache: Manage Files Across Sessions. 2019. Reference Source\n\nSmyth GK: Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol. 2004; 3(1): 3. PubMed Abstract \| Publisher Full Text\n\nSoneson C, Love MI, Robinson M: Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences [version 2; peer review: 2 approved]. F1000Res. 2015; 4: 1521. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWickham H, Averick M, Bryan J, et al.: Welcome to the tidyverse. J Open Source Softw. 2019; 4(43): 1686. Publisher Full Text\n\nXie Y: Dynamic Documents with R and Knitr. 2nd ed. Boca Raton, Florida: Chapman; Hall/CRC. 2015. Publisher Full Text\n\nXie Y: Knitr: A General-Purpose Package for Dynamic Report Generation in R. 2019. Reference Source" }	[ { "id": "60092", "date": "10 Mar 2020", "name": "Mark Dunning", "expertise": [ "Reviewer Expertise Bioinformatics", "Bioconductor", "Functional Genomics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nLee et al. present a case study of using Bioconductor packages to recapitulate an existing analysis. The analysis is performed without the need to worry too much about genome versions and using tools from tidyverse which were previously incompatible with standard Bioconductor methodologies. I see this being a useful reference for experienced users on how to run a modern RNA-seq analysis and perform data integration.\nI am pleased to say that I could re-run the code and reproduce the results presented. I can think of a few potential changes to increase its utility for beginner Bioconductor users.\nThe article assumes quite a lot of knowledge of tidyverse and GenomicRanges to understand the code chunks. Some pointers to where people can get more help on these packages would be useful. Some code chunks explicitly call methods from dplyr (dplyr::mutate), but not all. It would be good to be maintain consistency when calling dplyr methods, as the user would know where to get help.\nThe authors go straight from creating the DESeq dataset to running the differential analysis. In reality, one would never do this without performing adequate QC checks. I'm sure the authors must have done this (as indeed the original study authors must have). But showing the code to generate a PCA would be useful; along with interpretation of the results\nThe code is shown to remove genes with low counts prior to the DESeq analysis. I thought this was no longer necessary as DESeq2 incorporates it's own filtering. Or is this a memory-saving exercise? Some justification of why 10 counts and 6 samples was chosen would be beneficial to those new to Bioconductor and RNA-seq.\nThe authors use Genomic ranges to facilitate the integration of different assay types. Whilst it is critical to keep the data in this form for analysis, ultimately collaborators will want the results in spreadsheet format with recognisable gene names. It would be useful if the authors could include an example code snippet to annotate the overlapping regions (e.g. with gene symbols) and exporting as a csv.\nIt is my understanding that the authors present a simpler approach than that of Alasoo et al (2018)1 in order to illustrate the utility of Bioconductor infrastructure. Nevertheless, some commentary on whether the same results and biological insights are revealed by their approach would be beneficial.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "61441", "date": "30 Apr 2020", "name": "PuXue Qiao", "expertise": [ "Reviewer Expertise Statistics", "bioinformatics", "genomics", "transcriptomics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nLee, Lawrence and Love present a workflow article about using a group of well-constructed Bioconductor packages for fluent genomic analysis that integrates both RNA-seq and ATAC-seq analysis results from the same samples (baseline and stimulus).\nThe workflow provides instructions for obtaining differentially accessible (DA; peaks from ATAC-seq) and differential expression (DE; from RNA-seq) data objects, and more importantly a convenient way of integrating the ranged genomic features produced, while taking care of matching reference genome builds and other crucial “bookkeeping” tasks in the process.\nThe authors have demonstrated analysis can be performed to check whether DA peaks are enriched in the vicinity of DE genes guided by the principle that transcriptomic response to IFNg stimulation may be mediated through binding of regulatory proteins to accessible regions, and these bindings can be detected by ATAC-seq if they increase the accessibility of these regions.\nThe following steps are involved in the workflow:\n1. Downloading peaks data and RNA-seq data 2. Perform DE and DA analysis 3. Integrate ranges of DE and DA 4. Test enrichment of DA in DE gene vicinity versus non-DE genes with various thresholds\nMajor comments:\nThe process of 'resampling' subsets of non-DE genes from the pool of non-DE genes should be clarified to explain the purpose of doing such 'resampling'. In particular, whether it can be considered 'bootstrapping' should be addressed. Our understanding is that the resampling here is done without replacement from DE results, whereas for bootstrapping we expect to sample data records with replacement and recompute statistics of interest from them. Interpretation of the enrichment results or more discussion on the caveats of doing enrichment analysis like this are required.\nMinor comments:\nThe workflow was built with R-3.6.1 which results in a discrepancy with the current package's requirement of R >= 4.0.0. However, this is understandable since we are near the new release of Bioconductor but be reminded to re-compile the workflow with released version. A code chunk for checking that all required packages have been installed prior to other analysis in the environment would make the execution smoother for a user wanting to run the workflow. The downloading function `cache_atac_se()` raised an error when running on Windows. It can be fixed by setting mode = \"wb\" explicitly in the `download.file() `. Figure 1 which summaries the workflow could be improved by incorporating colored blocks for each package module and more descriptive elements companying the actual function calls or data objects. The caption also needs to provide more complete description of the elements of the figure for it to be comprehensible. These changes will give readers who are not familiar with all these packages more information. The optional code chunks (such as the Importing ATAC-seq data as a SummarizedExperiment object) could be moved down to appendix to make it clearer to the readers which things are core parts of the workflow and which are included for full transparency/reproducibility/data provenance. As for calculating the relative enrichment of peaks in DE versus non-DE, the description \"For all variables except for the origin column we divide the first row’s values by the second row,\" was inaccurate and could corrected to \"divide the DE row with the non-DE row\" It might worth emphasizing more on the contribution of this work in the abstract and introduction, maybe by stating explicitly the most attractive aspect of this work. For example, a guideline on integrating the ranged genomic features. Make the sub-headings consistent. Importing ATAC-seq, Import data. Some minor comments: library(fluentGenomics) appeared three times and library(plyranges) twice in the workflow. If this was to avoid namespace clashes, the function could be called explicitly plyranges:: instead of loading the packages multiple times? You could just use limma for the DE analysis as well and save yourself the loading of the DESeq2 package (joking!)\nIs the description of the method technically sound? Partially. The “resampling” procedure needs a bit more explanation such as clarifying how and whether this is considered a bootstrapping method.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "59959", "date": "21 May 2020", "name": "Mickaël Mendez", "expertise": [ "Reviewer Expertise computational genomics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe a workflow to perform exploratory analysis of biological data. This paper also highlights the importance of using tools that facilitate the application of essential concepts of exploratory data analysis: reproducibility and code readability.\nMajor comments:\n1. I was not able to successfully complete the protocol myself, failing at the second step. I tried three different environments:\n- R 3.4.4 on Ubuntu 18.04.4 LTS/Windows Subsystem for Linux WSL1/Windows 10 1909 18363.836 (BiocManager refuses to install fluentGenomics on the system R for what is currently the latest version of Ubuntu available for general availability Windows users). - R 4.0.0 x86 on Windows 10 1909 18363.836\n\n- BiocManager::install(\"fluentGenomics\")\n\n- BiocManager::install(\"sa-lee/fluentGenomics\")\nR version 4.0.0 (2020-04-24) -- \"Arbor Day\" Copyright (C) 2020 The R Foundation for Statistical Computing Platform: i386-w64-mingw32/i386 (32-bit)\nR is free software and comes with ABSOLUTELY NO WARRANTY. You are welcome to redistribute it under certain conditions. Type 'license()' or 'licence()' for distribution details.\n\nNatural language support but running in an English locale\nR is a collaborative project with many contributors. Type 'contributors()' for more information and 'citation()' on how to cite R or R packages in publications.\nType 'demo()' for some demos, 'help()' for on-line help, or 'help.start()' for an HTML browser interface to help. Type 'q()' to quit R.\n> library(fluentGenomics) > path_to_se <- cache_atac_se() Error in retrieve_cache() :\n\nPlease install rappdirs to set cache directory > detach(\"package:fluentGenomics\", unload=TRUE) > BiocManager::install(\"sa-lee/fluentGenomics\") Bioconductor version 3.11 (BiocManager 1.30.10), R 4.0.0 (2020-04-24) Installing github package(s) 'sa-lee/fluentGenomics' Error: package 'remotes' not installed in library path(s)\n\nC:/Users/mhoffman/R/win-library/4.0\n\nC:/Program Files/R/R-4.0.0/library install with 'install(\"remotes\")' > install.packages(\"remotes\") […] > BiocManager::install(\"sa-lee/fluentGenomics\") > path_to_se <- cache_atac_se() Error in retrieve_cache() :\n\nPlease install rappdirs to set cache directory > sessionInfo() R version 4.0.0 (2020-04-24) Platform: i386-w64-mingw32/i386 (32-bit) Running under: Windows 10 x64 (build 18363)\nMatrix products: default\nlocale: [1] LC_COLLATE=English_United States.1252 LC_CTYPE=English_United States.1252\n\nLC_MONETARY=English_United States.1252 [4] LC_NUMERIC=C\n\nLC_TIME=English_United States.1252\n\nattached base packages: [1] stats\n\ngraphics grDevices utils\n\ndatasets methods\n\nbase\n\nother attached packages: [1] fluentGenomics_0.99.3\nloaded via a namespace (and not attached): [1] Rcpp_1.0.4.6\n\nBiocManager_1.30.10\n\ncompiler_4.0.0\n\npillar_1.4.4\n\n[5] GenomeInfoDb_1.24.0\n\nXVector_0.28.0\n\nremotes_2.1.1\n\nbitops_1.0-6\n\n[9] tools_4.0.0\n\nzlibbioc_1.34.0\n\ntibble_3.0.1\n\nlifecycle_0.2.0\n\n[13] lattice_0.20-41\n\npkgconfig_2.0.3\n\nrlang_0.4.6\n\nMatrix_1.2-18\n\n[17] DelayedArray_0.14.0\n\nparallel_4.0.0\n\nGenomeInfoDbData_1.2.3\n\nrtracklayer_1.48.0\n\n[21] dplyr_0.8.5\n\nhms_0.5.3\n\nplyranges_1.8.0\n\nBiostrings_2.56.0\n\n[25] S4Vectors_0.26.1\n\nvctrs_0.3.0\n\nIRanges_2.22.1\n\nstats4_4.0.0\n\n[29] grid_4.0.0\n\ntidyselect_1.1.0\n\nglue_1.4.1\n\nBiobase_2.48.0\n\n[33] R6_2.4.1\n\nXML_3.99-0.3\n\nBiocParallel_1.22.0\n\nreadr_1.3.1\n\n[37] purrr_0.3.4\n\nmagrittr_1.5\n\nRsamtools_2.4.0\n\nmatrixStats_0.56.0\n\n[41] ellipsis_0.3.1\n\nBiocGenerics_0.34.0\n\nGenomicRanges_1.40.0\n\nGenomicAlignments_1.24.0\n\n[45] assertthat_0.2.1\n\nSummarizedExperiment_1.18.1 RCurl_1.98-1.2\n\ncrayon_1.3.4\n\nMinor comments:\n2. It is unclear who the audience for this paper is. As written it seems to presume a lot of technical knowledge about Bioconductor, R, and specific Bioconductor packages. It would not make a good starting point for someone who doesn’t have large familiarity with all of the above. This is too bad because it has the bones of something that could be useful to people with less expertise if some additional explanatory text were provided and clearer choices made about naming variables. There is also a danger that this will be used as a cookbook without understanding.\nI have marked some examples of things that could use more explanation but this is non-exhaustive. It would be worthwhile for the authors to examine the manuscript again and ask whether all of the large number of concepts, jargon, and names introduced are explained.\n3. The goal of the paper appeared to be clear after reading the paper. The introduction, figure 1, and following sections, however, leave the impression that the authors tried to reproduce results from another publication which is not the case. The paper would benefit from clarifying this toward the end of the introduction.\n4. Abstract: it is not clear which problem does the workflow solve and why is it important to solve it. For example, how does the import, model, and integrate structure increases result reproducibility? Is this structure new? Why would someone use it? We think that the abstract would benefit from more justification and moving the text specific to the tools used (tximeta, SummarizedExperiment) to the introduction.\n5. Abstract: The sentence “Using tximeta, ...” and the following can be confusing. Why is it only using of RNA-seq in the first sentence, and both RNA-seq and ATAC-seq in the following one?\n6. p3: Figure 1: The meaning of some of the objects is not immediately apparent. For example: coldata, se, dds, boot_genes. This figure does not make a great introduction for someone who is not already familiar with this workflow because so many of the details are mystifying. You should either simplify or explain more.\n7. p3: Figure 1: Text in boxes is blurry.\n8. p3: meaning of “fluent” should be described briefly here and not assumed\n9. Introduction: This section would benefit from a more thorough description of the tools used in the workflow and explain why they are important. For example: What is Bioconductor? Why is plyranges relevant for this analysis? What are tidyverse design principles and why are they relevant here?\n10. Introduction: Authors should also explain in the introduction why the “import, model, integrate” structure benefits users.\n11. The authors should consistently use the same tense (we examine … we will …).\n12. Why ignoring genotypes?\n13. It would be useful to note that \"genomic ranges\" are often referred to as intervals elsewhere such as in BEDTools.\n14. Please cite Tximeta at the first occurrence.\n15. Experimental data: Here and in the following sections, the authors describe alternatively what the workflow does and what the workflow can do. For example: “The following code loads the path to the cached data file, or if it is not present, will create the cache and generate a SummarizedExperiment using”. We think that it would be clearer if these sections with code examples focus on what exactly the code does, like in a protocole paper. Describing what the tools can do should appear either in the introduction or in a separate paragraph. For the example above, the paragraph would explain how users would benefit from this behavior: save time?\n16. “A subset of the RNA-seq ...”, Why use only a subset?\n17. Typo: “be also be downloaded”\n18. p4: “GENCODE” not “Gencode”\n19. p4: Specify whether basic or comprehensive gene set used\n20. p4: Unclear what “Importing ATAC-seq data as a SummarizedExperiment object” refers to\n21. Import data as SummarizedExperiment: Authors should briefly describe what methods Soneson et al. use to summarize transcripts at gene level, and justify why they are doing it.\n22. p4: Unclear where the `coldata.csv` file is or what that name refers to. “file” should not be in monospace text\n23. p4: Placement of “Wickham et al., 2019” makes it appear to be a citation for R rather than tidyverse.\n24. p5: Many things here unclear to the uninitiated: what is the scary message when you load dplyr? What is a factor and relevel? What is a tibble? What do the ~ mean?\n25. p5: Meaning of “but would typically not have the ‘gz’ ending if used from Salmon directly” unclear\n26. p5: Should note the “files” column is going to be different for different users\n27. p6: What is suppressPackageStartupMessages?\n28. p6: What is a Salmon gtf?\n29. p6: Lots of unexplained details in the two transcripts on this page.\n30. p7: “tximeta knows the correct reference transcription”. How?\n31. p7: GRanges not described or cited\n32. p7: Unclear why only the start of + genes and not also their end is affected\n33. p7: The sentence starting “Another alternative would be” is complex and its meaning unclear (alternative to what?)\n34. Last paragraph of \"Import data as SummarizedExperiment\": authors describe alternative ways of summarizing genes and using their representative TSS. Is the point to explain that GRanges make these operations easier? We think a potential user would like to know how to perform precisely these operations. Otherwise move this to an earlier paragraph describing what the tool is capable of doing.\n35. p7: What does “109M” refer to? Please use SI or IEC units here and elsewhere\n36. p7: Should be “370 MB” or “370 MiB” not “~370 Mb”\n37. p7: It’s not clear how to actually get the tab-delimited files, a departure from what is otherwise detailed step-by-step instructions\n38. p7: Why skip=1? Why [,-1]?\n39. p7: It would be better if instead of just saying “we know this from the Zenodo entry” you instruct the workflow user to check the Zenodo entry to find out what genome assembly is used. A screenshot of that part of the Zenodo entry might be helpful.\n40. p7: Citation for Genome Reference Consortium needed\n41. p8: what is col_types? How do you know that this is what the col_types should be?\n42. p8: This is the first instance of a convention of using comments in monospace text for explanatory notes. I would avoid this and stick to notes in proportional text.\n43. p8: what is a “nominal” FDR?\n44. p8: greater than 1 or greater-than/equal? Suggest using > or >= for greater clarity\n45. Why 1%?\n46. p9: “MA” needs expansion, citation, and ideally brief explanation\n47. p9: Figure 2 caption has insufficient detail. Should describe what each point represents, and how many points there are. Should describe what method is used to adjust p-values. Please describe triangles versus circles. Explain the red horizontal line. Please indicate that “log fold change” means log2. Describe what “normalized counts are”\n48. p9: “due to the convention of plyranges”—meaning unclear\n49. p9: “This kind of book-keeping” = ?\n50. p9: Not everyone reading this will work on human genomics and know that hg38 = GRCh38\n51. p9: The authors should consistently put explanations of transcripts either below the transcript or above and not switch back and forth. (I think below is probably more helpful.)\n52. p10: please make clear that the altHypothesis here gets ALL of the other genes, that there are no problems with the edge case at LFC = threshold\n53. p11: Lots unexplained in the limma transcript\n54. p12: What is a “seqlength”?\n55. “genes without strong signal of DE”, are these “non-DE” genes? Would be better if used consistently.\n56. p12: Down sampling -> “Downsampling”\n57. p12: Unclear why one would want to downsample. The description only mentions that one can.\n58. p13: Please explain why one would want to set a seed.\n59. p13: “This creates a list of _GRanges_ objects as a list”. It would be clearer to say that this creates a _list_ of 10 _GRanges_ objects.\n60. What are \"verbs\" for GRanges\n61. p13: The importance of doing the bootstrap procedure here is really unclear\n62. p14: Why is there a circular sequence? I assume this is the mitochondrial chromosome? Does its presence in the background disturb the analysis at all?\n63. p14: \"Now we would like to modify our gene ranges so they contain…\" Why?\n64. p14: should be 5 prime not 5 apostrophe\n65. p15: the significance of the clarification that each “gene” is just a tuple of chromosome, start, end, strand is unclear\n66. p15: A lot of na.rm or is.na from here outward. Would be worth explaining why this is.\n67. p16: Figure 3: This figure needs descriptive labels instead of references to variables used in the code.\n68. p19: Explain why there are missing values\n69. p20: Should comment on why Figure 4 is non-monotonic\n70. p20: Saying the x-axis is on a log10 scale is a bit confusing because the y-axis is also on a log10 scale. It is better to make log scales more obvious by having non-uniform tick marks and grid lines. Also I don’t think the x-axis is on a log10 scale, log fold change here is measured with log2. (This would be more obvious if in the text you called it \\log2 fold change consistently instead of the ambiguous “LFC”.)\n71. p21: It is unfortunate that the main point of this paper is to show that you can do something “fluently”, while what that means is never discussed or justified in the paper.\n72. p21: “There are several further steps that would be interesting to perform”, Are these steps easy to implement with the tools you described? If so, can you explain how easier it is with your approach?\n73. p21: “Finally, our workflow illustrates the benefits of using appropriate data abstractions”, This is probably obvious for R power users but not for other readers. Can you point at a specific section that shows that a user will benefit from using your approach?\n74. p21: The data availability statement is confusing. Clearly not all data are part of the article, they are part of external packages or articles (some specified without a particular URL or accession number, just reference to a paper).\n75. p21: instructions for installing BiocManager would be helpful, as would listing the minimum version of R or any other relevant packages. Also should mention that you need to library(BiocManager) first\n76. p21: It is unclear why you would want the development version, especially since it seems to be an older version number than what’s on Bioconductor\n77. p23: Items in the bibliography are not consistently referenced with specificity in a visible way. DOI should be provided for all Zenodo entries not just some. Some other documents that are not referenced via traditional publishing (journal volume and page number, or monograph from a publisher) should probably have DOI or URL specified also.\nDiscretionary recommendations:\n78. p4: `path_to_se` is a poor name for a variable. It is not orthogonal to the other variable names used here and has little explanatory power. I recommend `atac_filename`\n79. p4: `dir` also is not descriptive. I recommend `quant_dirname`.\n80. p4: Would suggest more descriptive names than colfile/coldata, orthogonal to whatever naming scheme you use (which means either changes to names here or to use variations of the names I used above)\n81. p5: This transcript is a lot to digest, and it’s hard to know which parts of the explanatory text refer to which part of the transcript, which has multiple commands that go back a page previous. I suggest breaking it up more with explanatory text to the extent possible. Piping is convenient for a programmer but not for explanation. This comment applies to many of the subsequent transcripts too\n82. p5: Get the display of coldata so that it doesn’t exceed one line per row\n83. p6: It’s not a good idea to call your experiment object merely `se` when it is one of multiple SummarizedExperiments here\n84. p6: Please use a consistent style for spacing around = in R code\n85. p7: I suggest not giving the same name to different objects (`atac_mat` is first something like a data.frame and then a matrix.\n86. p7: Suggest not shadowing built-in functions with variable names like `rownames`.\n87. p7: “We know this” antecedent unclear\n88. p8: I suggest running all the R code in here through a linter. This will ensure you have consistent style.\n89. p8: res is not a specific variable name\n90. p10: Why switch between “other genes” and “non DE genes”? Just call them `not_de_genes` from the start. (Or switch to `genes_de` and `genes_not_de`.)\n91. p11: Why is library(fluentGenomics) loaded again?\n92. p11: Using library(fluentGenomics) adds a data object called `peaks` to your environment? This seems undesirable\n93. p12: The text would be a bit more readable if you eschewed the abbreviations “DA”, “DE”, and “LFC” in the main text.\n94. p13: here and elsewhere: integer literals (e.g. 10L) should be used instead of float literals for integers\n95. p14: why `2*1e4`? 20000L would be clearer\n96. p16: Figure 3: Please specify what the components of the box plot mean. There is no universal box plot.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? No\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-109
https://f1000research.com/articles/9-108/v1	11 Feb 20	{ "type": "Research Article", "title": "Frequency and associated factors related to sexual addiction in medical students from 16 Latin American cities, 2016: a regional multicentric study", "authors": [ "Christian Richard Mejia-Alvarez", "Jhosselyn Chacon-I", "Dayanne Benites-Gamboa", "Niels Pacheco-Barrios", "Giancarlo F. Castillo-Tarrillo", "Dercy Centeno-Leguia", "Joseph Wendell-Cubilla", "J.Antonio Grandez-Urbina", "Christian Richard Mejia-Alvarez", "Jhosselyn Chacon-I", "Dayanne Benites-Gamboa", "Niels Pacheco-Barrios", "Giancarlo F. Castillo-Tarrillo", "Dercy Centeno-Leguia", "Joseph Wendell-Cubilla" ], "abstract": "Background: Examples of addiction problems that have been reported in growing populations are those related to sexual impulses and addictions. However, such studies have not been carried out in Latin America. The aim of this study was to characterize and identify possible associations of sexual addiction in medical students in Latin America. Methods: An analytical cross-sectional study was carried out among the university students of a medical school in 16 cities; students of medical schools were interviewed during the first semester of 2016. To define sexual addiction, the multi-cage cad-4 test was used, categorizing individuals as possibly or not a potential problem. Additionally, associations with several social and educational variables were obtained. Results: In our study, 6% (221) of the 3691 respondents exhibited a possible problem of sexual addiction; men had 95% more problems (95% confidence interval (95%CI): 21-214, p=0.006), for each year of age it increased by 9% (95%CI: 1-18%, p=0.034 ), those who had a partner were 67% more likely to exhibit sexual addiction (95%CI: 1.34-2.08%, p <0.001) and those who professed a religion present 44% less frequency (95%CI: 20-60%, p: 0.001). When adjusted for marital status, having children, year of studies, and the university where the respondent studied were not associated. Conclusion: Although the percentage of students who had problems with sexual addiction is minimal, screening programs should be created to find students who suffer from these problems, to avoid the possible consequences that may arise.", "keywords": [ "Sexual adiction", "risk factor", "medical students", "sexual behavior" ], "content": "Introduction\n\nThroughout life, young people experience a series of changes, including biological, cognitive and psychosocial type changes, which are influenced by various factors, such as gender, social, cultural, political and economic environment, as well as the level of physical and psychosocial/cognitive maturity of the person1. Some studies have found that by submitting these young people to stressful environments can trigger them to be disinhibited, which leads them to adopt risky behaviors1,2. It is essential to know that one of the populations with the most considerable stress and interpersonal relationships is the students3. If early sexual initiation and the non-use of protective measures are added to this, it becomes an immediate health problem4, especially if these individuals have had an inadequate sexual education5.\n\nSome studies have explored the sexual sphere of university students, for example, in Turkey, this topic was characterized, and other topics considered “taboo” for their culture were touched6. In Finland, thousands of university students were surveyed to determine continuity/frequency according to age group7, another one performed in India measured sexual knowledge and practices8. All of these studies highlight the importance of the student having an adequate orientation for the correct expression of their sexuality. In the future, this will help them to advise their patients, allowing them to establish better interactions and communication with them9. In Latin America, the studies performed in the sexual sphere try to link the sexual behavior with factors that could influence this behavior, finding only one study, performed in Colombia, that analyzes this relationship through the lifestyle of university students10. For all these reasons mentioned, the objective of the study was to characterize and find the associations of sexual addiction that medical students have in Latin America.\n\n\nMethods\n\nWe performed an analytical cross-sectional study; this was based on the data that was generated after the application of surveys to medical students from 16 cities in Latin America.\n\nThe students resided in the cities of David, Panama; San José, Costa Rica; Tucumán, Argentina; Ciudad Bolivar, Venezuela; Tunja, Colombia; Bogotá, Colombia; Cochabamba, Bolivia; Tarija, Bolivia; Arequipa, Peru; Metropolitan Lima, Peru; Ayacucho, Peru; Ica, Peru; Chiclayo, Peru; Cusco, Peru; and Huancayo, Peru.\n\nA multicenter study was conducted, because the minimum sample size required was 3506 medical students, this is important to find minimum differences of 1% of sexual impulses according to marital status (the smallest difference detected in the prior study); this was calculated with a potency of 80% and a confidence level of 95%. Calculation was performed using STATA 11.1 with the SAMPSI command for sample size calculation,\n\nWe calculated the statistical power for the crossing of the variables; in almost all cases, the statistical power obtained was greater than 99%. However, in two of the crosses, the power was not reached (for marital status: 56% and for the type of the university: 25%); Therefore, the results obtained in these two cases must be taken as purely exploratory.\n\nMedicine students of both sexes who agreed to participate voluntarily between 18 and 26 years in the study were included, they were recruited by direct contact. Written informed consent was obtained, and 148 surveys that had no answers from the primary survey were excluded, from which the dependent variable was extracted.\n\nThe study began in April 2016, after the Ethics Committee of “Hospital Nacional Docente Madre Niño San Bartolome” approved the research with the code CIEI-170023, and was completed in April 2017. The study was performed by capturing the necessary amount of medical schools to reach the minimum sample size; those in charge of surveying in each campus made the request for institutional permission.\n\nWe explained the purpose of the study and that the participation was voluntary and anonymous; The students were surveyed in environments where students had the necessary privacy conditions for the proper resolution of the survey.\n\nThe primary variable was based on a previously validated questionnaire11. The CAD-4 Multi Cage Test used consist in the validation of eight kind of addiction, so we based our questionnaire in four questions related to sexual addiction:\n\n1. Has your sexual activity prevented you from performing usual tasks in your life, such as work or family obligations?\n\n2. Have your partners complained about your excessive sexual activity?\n\n3. Have you ever considered that your sexual activity is excessive?\n\n4. Have you ever tried unsuccessfully to moderate your sexual activity?\n\nFor this investigation, only the questions that measured sexual addiction were taken. The questionnaire is self-administered and is answer on a dichotomous scale (yes/no) and it is estimated that none or an affirmative answer indicates not an existence of that problem; two affirmative answers indicate a possible presence of that problem; three affirmative answers suggest a very probable presence of that problem; and answer affirmative at four suggests the sure existence of that problem11. The cut-off point was two definite answers, as an answer of zero or one indicated the absence of this problem. On the other hand, two or more positive answers indicated there was the possible existence of addiction. Frequencies and percentages were described. Also, this was disclosed according to the number of affirmative answers that all surveyed students had.\n\nThe dependent variable was crossed with the independent variables using the chi-square statistical test for crossing with sex, marital status, having a romantic partner currently, having children, and studying in a private university. The dependent variable was compared to the quantitative variables (age and year of studies) with the sum of Wilcoxon ranks. The software used to perform the analysis was STATA 14.0.\n\nA 95% confidence level was used for bivariate and multivariate analysis, prevalence ratios (RP), 95% confidence intervals (95% CI) and p values (considering values p<0.05 as statistically significant), through the use of generalized linear models, with the Poisson family, the log link function, robust models and using the university as a cluster adjustment group. For the preparation of the multivariate model, all the variables in Table 1 were taken into account (both those that were statistically significant and those that were not, this being important in the context of a student with a possible addiction to sex). The variables that were significant in the bivariate analysis were maintained in the multivariate. Of those that were not significant, age became significant in the multivariate model (the others served as an adjustment).\n\n* Medium and interquartile range. P values obtained with chi square (for two categorical variables) or sum of ranges (for the cross of a quantitative variable versus a categorical variable).\n\n\nResults\n\nA total of 3691 students were surveyed in the medical schools, according to the answers of the cad-4 multi cage test, it was obtained that 87.3% (n=3222) and 6.7% (n=248) had no positive answers and had only one positive answer regarding sexual addiction, respectively. These individuals were classified as having no sexual addiction. In total, 3.1% (n=113), 1.9% (n=70), and 1.0% (n=38) had 2, 3, and 4 definite answers, respectively. These individuals were categorized with sexual addiction problems. Table 1 shows the characteristics of the students determined to exhibit potential sexual addiction problems. De-identified responses for each participant are available as Underlying data12.\n\nFigure 1 shows the percentage of sexual addiction according to the year of study of the surveyed students. Students residing in the capitals of Peru and Colombia had the highest frequency of sexual addiction problems, both with 11%, while those who studied in the cities of Tucumán, Argentina and Ciudad Bolivar, Venezuela both had 0% (Figure 2).\n\nIn the multivariate analysis, an association was found between the highest frequency of sexual addiction and male sex (PR: 1.95; 95% CI: 1.21-3.14, p-value: 0.006), years of age (PR: 1.09; 95% CI: 1.01-1.18; p-value: 0.034) and having a partner (PR: 1.67; 95% CI: 1.34-2.08, p-value <0.001).\n\nOn the other hand, those who described themselves as followers of a religion had a lower frequency of sexual addiction (PR: 0.56; 95% CI: 0.40-0.80; p-value: 0.001); all these variables were adjusted for marital status, having children, the year of studies and using the university where the surveyed studied as a cluster (adjustment variable) (Table 2). Figure 3 shows the percentages of sex addiction according to the statistically significant categorical variables in the multivariate model. Figure 4 shows the percentages of specific answers to the four questions of the sexual addiction test in medical students in Latin America.\n\nRP, reason for prevalence; 95%CI, 95% confidence interval. p-value obtained with generalized linear models, with Poisson family, log link function, robust models and using the university as a cluster group.\n\n\nDiscussion\n\nOur study found that few students had a sexual addiction. Our findings differ from a study performed at a private university in the US, which showed that 26% of the students had symptoms of risk for sexual addiction13; this could be due to population differences. Broader population studies have obtained similar percentages to ours. A nationwide study conducted in Sweden found that 10% of the population is considered \"hypersexual\"14. Regional and local surveys have been performed in the US, which suggests that approximately 5% of the general population can meet the criteria of a compulsive sexual disorder15. Another factor that led to finding different results could be the test that was applied, because some research is not based on validated instruments. These results should be confirmed, since the objective of the study was not to give prevalence, because a random sampling was not performed out in each campus, so each institution should rate this situation of its students.\n\nTt was found that men had higher frequencies of sexual addiction; this is similar to what was found in studies conducted in Mexico, where more male university students than female had a sex addiction and risky sexual behavior, such as practicing sex without condoms16. Therefore, counseling activities should focus on them so that they can receive recommendations for safe sex practices.\n\nWith regard to age, the frequency of sexual addiction increased at an older age; these results are consistent with studies performed in Sweden and the United States of America; in which it was determined that young adults are more likely to suffer from \"hypersexuality\" and to show behaviors related to compulsive sexuality14,17. However, it is essential to highlight that the relationship between younger age and hypersexuality has not always shown an association, being demonstrated by a study that used a virtual platform18. Therefore, it is necessary to characterize the age groups that present more significant problems of addiction in each reality, to focus on them the programs of prevention of sexually transmitted diseases.\n\nIt was also found that people who have a sexual partner had a higher frequency of sexual addiction problems; this is similar to what was found in an investigation in Mexico19. Those who professed to have a religion had lower frequencies of sexual addiction; this could be due to the less liberal life they present and the religious principles they have20.\n\n\nLimitations\n\nOur study had the limitation that there was no random sampling, which generated a selection bias. However, this is the first report of this problem in the student population, and because of the large number of students that were achieved, this generates results that are pertinent to the subject addressed.\n\n\nConclusion\n\nIt is concluded that sexual addiction problems occurred in a small proportion of Latin American medical students. These addiction problems were positively associated with male sex, age, and having a romantic partner, being negatively associated with professing a religion.\n\n\nData availability\n\nFigshare: Sexual_addiction_med_students_LATAM. https://doi.org/10.6084/m9.figshare.9917018.v212.\n\nThis project contains basic demographic information about each participant, alongside their answers to each of the questions asked.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nThanks to all those who gave their input for the elaboration and review of the work, thanks to these comments, the research won the second place in the scientific contest organized by the Latin American Federation of Scientific Societies of Medical Students, occurred in Bolivia during the month of September.\n\n\nReferences\n\nShutt-Aine J, Maddaleno M: Salud sexual y desarrollo de adolescentes y jóvenes en las Américas: Implicaciones en programas y políticas. OPS, Washington, DC. 2003. Reference Source\n\nLuquis RR, Brelsford GM, Rojas-Guyler L: Religiosity, spirituality, sexual attitudes, and sexual behaviors among college students. J Relig Health. 2012; 51(3): 601–14. PubMed Abstract \| Publisher Full Text\n\nGómez-Restrepo C, Rodríguez MN: Factores de riesgo asociados al síndrome depresivo en la población colombiana. Rev Col Psiqui. 1997; XXVI(1): 23–25. Reference Source\n\nSintes RA, Alonso GD, Mainegra IS, et al.: Temas de Medicina General Integral. 2 ed. La Habana: Editorial Ciencias Médicas; 2008: 148–56.\n\nAdolescencia y sexualidad [citado 2 Nov 2011]. Reference Source\n\nYazici S, Dolgun G, Zengin N, et al.: The Determination of University Students’ Knowledge, Attitudes and Behaviors on the Matter of Sexual Health. Sex Disabil. 2012; 30(1): 67–75. Publisher Full Text\n\nVirtala AM, Kunttu K, Huttunen TA, et al.: Sexual intercourse and current contraceptive use among university students in Finland. Eur J Obstet Gynecol Reprod Biol. 2007; 135(1): 104–10. PubMed Abstract \| Publisher Full Text\n\nAggarwal O, Sharma AK, Chhabra P: Study in sexuality of medical college students in India. J Adolesc Health. 2000; 26(3): 226–9. PubMed Abstract \| Publisher Full Text\n\nMudd JW, Siegel RJ: Sexuality--the experience and anxieties of medical students. N Engl J Med. 1969; 281(25): 1397–403. PubMed Abstract \| Publisher Full Text\n\nLema Soto LF, Rubio Sarria A, Salazar Torres IC, et al.: Comportamiento y salud de los jóvenes universitarios: satisfacción con el estilo de vida. Pensamiento Psicológico. 2009; 5: 71–87. Fecha de consulta: 28 de mayo de 2016. Reference Source\n\nPedrero EJ, Rodríguez MT, Gallardo F, et al.: Validación de un instrumento para la detección de trastornos de control de impulsos y adicciones: el MULTICAGE CAD-4. Trastor Adict. 2007; 9(4): 269–78. Publisher Full Text\n\nMejia CR, Jhosselyn CI, Dayanne BG, et al.: Sexual_addiction_med_students_LATAM.xlsx. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9917018.v2\n\nSeegers JA: The Prevalence of Sexual Addiction Symptoms on the College Campus. Sex Addict Compulsivity. 2003; 10(4): 247–258. Publisher Full Text\n\nLångström N, Hanson RK: High rates of sexual behavior in the general population: correlates and predictors. Arch Sex Behav. 2006; 35(1): 37–52. PubMed Abstract \| Publisher Full Text\n\nColeman E, Raymond N, McBean A: Assessment and treatment of compulsive sexual behavior. Minn Med. 2003; 86(7): 42–7. PubMed Abstract\n\nFlores YYR, Rangel MG: Influencia del rol de género en la conducta sexual de riesgo en adolescentes universitarios. Index Enferm. 2010; 19(4): 245–248. Publisher Full Text\n\nKafka MP, Hennen J: Hypersexual desire in males: are males with paraphilias different from males with paraphilia-related disorders? Sex Abuse. 2003; 15(4): 307–21. PubMed Abstract \| Publisher Full Text\n\nWalton MT, Cantor JM, Lykins AD: An Online Assessment of Personality, Psychological, and Sexuality Trait Variables Associated with Self-Reported Hypersexual Behavior. Arch Sex Behav. 2017; 46(3): 721–733. PubMed Abstract \| Publisher Full Text\n\nZapata J, Petrzelová J, Chávez M: Actitudes respecto a la sexualidad en estudiantes universitarios. Enseñanza e Investigación en Psicología. 2009; 14(1): 137–151. Reference Source\n\nVigil PP, Riquelme RR, Rivadeneira HR, et al.: TeenSTAR: una opción de madurez y libertad. Programa de educación integral de la sexualidad, orientado a adolescentes. Rev méd Chile. 2005; 133(10): 1173–1182. PubMed Abstract \| Publisher Full Text" }	[ { "id": "68735", "date": "02 Dec 2020", "name": "Mahmood Karimy", "expertise": [ "Reviewer Expertise public health", "addiction", "change behavior", "risk behavior" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe introduction is very short, please describe the importance, necessity and benefits of this study.\nThe main issue of the introduction is that the relevance of the study is not clear. First, the risks of existing sexual addiction in students are underrepresented . Second, it is unclear what the added value of this study is in the current field.\n\nAnother issue is that the first paragraph should state the definition of sexual addiction behavior. The definition was must conform DSM-IV or ICD-10.\nMethods:\nThe choice for administered on all questions is unclear. For example, why is there no researcher/teacher report on sexual behaviors of the student in the classroom? Also, strength in the design is the fact that the teacher left the classroom during completing the questionnaire, in the hope student answered the questions socially less desirable. However, it is unclear what was done when the students finished their questionnaire. How were the questionnaires collected?\n\nPlease write the inclusion and exclusion criteria.\n\nPlease add the used method for the sampling.\nResult:\nOK.\nDiscussion:\nThe generalization of the results should be stated.-The conclusion should be short aim the main objective of the study based on the study results.\n\nThe implications of the study are just marginally described. Preferably, given the similarities and differences between other international studies, which policy would be suitable for world students? And which group should be targeted in prevention plans, and which goals fits the results?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "77755", "date": "17 Feb 2021", "name": "Adesola Oluwafunmilola Olumide", "expertise": [ "Reviewer Expertise Adolescent health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral Comments The paper presents important findings. The authors however need to make revisions in line with suggestions made before it can be fully accepted for indexing. Importantly, the paper requires English language editing to improve comprehension by readers. The editing needs to be done before final approval.\nThe paper describes findings on information obtained from 16 different cities but this has been treated as one i.e. findings are not disaggregated by city. Could authors provide some explanation for why they decided to analyze and present their findings like this?\n\nWere all 16 cities in 16 different countries or were there situations in which more than one city was selected in a country?\nComments on specific sections of the paper\nIntroduction\nDelete “by” in the statement (Introduction; paragraph 1) below. \"Some studies have found that by submitting these young people to stressful environments can trigger them to be disinhibited, which leads them to adopt risky behaviors\".\n\nPlease revise the statements below. Meanings are not clear. In Finland, thousands of university students were surveyed to determine continuity/frequency according to age group7, another one performed in India measured sexual knowledge and practices8.\nMethods.\nSampling Please provide additional information about how the participants were recruited. Was this by simple random sampling or some other convenience??\n\nPlease revise the statement below. It is currently not clear: Statistical analysis: The dependent variable was crossed with the independent variables using the chi-square statistical test for crossing with sex, marital status, having a romantic partner currently, having children, and studying in a private university.\nDiscussion\nDiscussion Paragraph 1, lines 13 to 16, “These results should be confirmed, since the objective of the study was not to give prevalence, because a random sampling was not performed out in each campus, so each institution should rate this situation of its students”. The information is not clear.\n\nDiscussion paragraph 2, Opening line: “Tt was found that men …..” Please change Tt to “It”.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-108
https://f1000research.com/articles/9-107/v1	11 Feb 20	{ "type": "Case Report", "title": "Case Report: Late onset of generalized isomorphic morphea in a postmenopausal woman", "authors": [ "Marie Angelique Lazo-Betetta", "Renzo Perez-Vasquez", "Arantxa Sanchez-Boluarte", "Fiorella Inga-Berrospi", "J. Antonio Grandez-Urbina", "Marie Angelique Lazo-Betetta", "Renzo Perez-Vasquez", "Arantxa Sanchez-Boluarte", "J. Antonio Grandez-Urbina" ], "abstract": "Morphea is an inflammatory, sclerosing skin condition of unknown cause that generally does not present systemic manifestations. A 66-year-old Caucasian Peruvian female patient, who was previously a nurse, presented with a prior history of 4 years of indurated dermal plaque lesions with constant progression. Diagnosis of morphea was made by clinical examination and skin biopsy. The patient started topical treatment with methoxsalen and phototherapy. When no improvement was seen, it was switched to methotrexate. However, due to changes in liver profile, phototherapy was restarted with progressive clinical improvement. It is essential to differentiate all morphea subtypes for proper management.", "keywords": [ "Isomorphic generalized morphea", "indurated plaque", "postmenopausal woman" ], "content": "Background\n\nMorphea is a rare, sclerosing inflammatory condition of the skin that can involve underlying soft tissues and cause a severe cosmetic and functional defect of unknown etiology. Associated factors, such as genetic predisposition, immune dysregulation, and environmental factors, have been reported1,2. The current classification includes five subtypes: circumscribed, generalized, linear, pan-sclerotic, and mixed (Table 1)3,4. Generalized morphea has two patterns of cutaneous distribution, symmetric distribution and isomorphic distribution, which occurs almost exclusively in postmenopausal patients5,6. The annual incidence ranges between 3.4 and 27 cases per 1,000,000 people, and is more frequent in women than men. This disease has a peak of bimodal incidence, with the age of presentation being between 7 and 11 years in children and 44 and 47 years in adults7,8.\n\n\nCase presentation\n\nA 66-year-old postmenopausal Caucasian Peruvian female patient, who was previously a nurse of Swedish ancestry, with prior history of Type 2 Diabetes Mellitus and insignificant family history presented at an outpatient facility of an allergy and immunological center in Lima, Peru. She had a prior history of pathology biopsy report of morphea, she was treated with topical methoxsalen previously (with good outcome) in Sweden.\n\nThe clinical episode began four years ago, starting with indurated, painless skin lesions without erythema in the breast folds, which progressively increased locally. These lesions appeared in other regions, such as the hip and upper and lower extremities, presenting plaques with indurated hiccups and hyperpigmentation (Figure 1–Figure 3). The patient was diagnosed with morphea following clinical examination and a prior pathological biopsy, which reported morphea, in Sweden. A timeline of care is available in Figure 4.\n\nInitially, pharmacological treatment was initiated, we use topical treatment with methoxsalen (0.1% lotion applied 30 minutes before phototherapy) and phototherapy (light therapy using artificial ultraviolet A light) the wavelength was 315–400 nm with 3.10–3.94 eV photon energy in lesions five times a week for six months. The patient had good adherence to the treatment. However, no improvement was seen, so the treatment was changed to methotrexate administrated orally (7.5 mg twice a week). Measuring the tolerance of the pharmacological treatment with liver profile (aspartate and alanine transaminases and total, indirect and direct bilirubin were measured). After six weeks, the patient exhibited alterations in liver profile, this being an expected adverse event. Therefore, the medication was discontinued, after which phototherapy was restarted twice a week, without topical treatment. Visible improvement of the lesions was achieved, the upper limbs being the location that was initially remitted. Subsequently, the frequency of phototherapy went from twice a week to once a week, for two years (Figure 1).\n\nCurrently, the patient has inactive lesions with the presence of dystrophies and atrophic skin, with annual follow-up controls to assess for disease remission and a good prognosis. The patient has a follow-up period of two years after last course of treatment and is seen every year.\n\n\nDiscussion\n\nMorphea is a rare disease, with an estimated incidence of 27 cases per million people. It is suggested that it has an autoimmune origin. In relation to pathophysiology, little is known until now. However, some risk factors have been proposed, the most reported being: trauma, radiation, Borrelia burgdorferri infection, and certain medications9,10. The age of presentation of this disease in childhood and adulthood, is the age range of 4–62 years, with a mean age of presentation of 35–37 years; in addition, it is more frequent in women than in men, with a ratio of 3:1.9,11. The presentation is characterized by erythematous or violaceous skin lesions, with the progression of the disease sclerotic plaques develop in the center of these lesions. In some patients, one of the predominant characteristics is the development of hyperpigmentation, and sclerosis may be limited or absent7,12. Generalized morphea is characterized by superficial, long, and coalescing plaques in multiple parts of the body. This type of morphea is more common in adults than in children, and sclerosis usually occurs in the trunk, upper and lower limbs, with preservation of the face, hands, and feet7,13.\n\nRegarding the complications of this disease, the injuries caused to the skin can generate functional deterioration and limitation of the motility range of the affected area. The disease can also produce arthralgia, edema, and contractures in the extremities. Initially, sclerotic plaques are formed, which after months and years become hypopigmented or hyperpigmented atrophic plaques that manifest as a depressions in the skin14,15. In patients with compromised dermis, subcutaneous tissue and muscle, squamous cell carcinoma may be formed due to the present of chronic inflammation16,17.\n\nIn many patients, the diagnosis can only be achieved using clinical findings. However, some imaging studies such as ultrasound, 2-D shear wave elastography and magnetic resonance imaging would be useful in assessing and quantifying morphea extension. In other and histopathological changes, such as compromised dermis, subcutaneous tissue and muscle, may be useful to confirm the diagnosis and evaluate the extent of the disease. Skin biopsy can provide additional information in those patients where the physical examination is inconclusive, also allows us to have information on the depth of the condition and the activity of the disease. Walker et al. assessed a cohort of 83 adults and children with morphea, where they sought to systematize morphological changes and determine the association between histopathological and clinical findings18. In total, 91 biopsies were performed in 83 patients, and they found three patterns of dermis involvement: superficial, deep, and total sclerosis, which can occur in the different morphea subtypes. For example, in the generalized symmetric morphea, a pattern of deep affection of the dermis predominates; in the generalized isomorphic morphea, a pattern of superficial affection is often presented; on the other hand, lichen sclerosis morphea involves a pattern of linear and superficial affection. In this case, after having carried out a detailed study, skin biopsy was performed, which confirmed the diagnosis of morphea18.\n\nDharamsi et al.19 conducted a cohort study of 181 patients vs controls with the objective of determining the prevalence of antinuclear antibodies, anti-histone antibodies and anti-single-strand DNA antibodies finding that the prevalence of morphea is 34% antinuclear antibodies, 12% anti-histone antibodies and 8% anti-single-strand DNA antibodies. Concerning the patient discussed in this case study, we did not have the results of autoantibodies, so it is not possible to stratify it in any of the groups mentioned above.\n\nThe differential diagnosis for morphea is usually systemic sclerosis, this being a systemic disease characterized by progressive dermal sclerosis with the involvement of internal organs. The typical cutaneous manifestations are evidenced symmetrically at the proximal level, presenting as sclerodactyly, Raynaud’s syndrome, facial, trunk and/or limb sclerosis with significant functional limitation, sometimes accompanied by pulmonary and cardiac involvement. Regarding the patient, the condition was exclusively cutaneous without systemic manifestations and without compromising the proximal part of the hands, as happens in systemic sclerosis, also taking into account that the condition is currently in remission without treatment20.\n\nIt should be taken into account that the patient was previously diagnosed and had received treatment in Sweden. The patient therefore had access to an advanced health system and did not encounter present economic limitations; it was possible to have timely diagnosis and treatment, which positively affected prognosis. However, within the limitations of the study, it was not possible to have access to the patient’s complete medical history; only the information provided adequately by the patient was used. At present, there are various treatment options for active lesions in generalized morphea, whether superficial or deep21. Concerning superficial lesions, it is possible to initially use phototherapy as the first line of treatment throughout the body22. If phototherapy is ineffective, a systemic treatment that includes methotrexate or mycophenolate, alongside systemic glucocorticoids, should be used23. If the lesions are deep, immediate use of systemic treatment is recommended and phototherapy should be avoided due to poor penetration of ultraviolet light in the deep tissues24. There is also topical therapy in which corticosteroids, vitamin D, and tacrolimus are used. However, it has not proven to be useful in cases of generalized morphea25.\n\n\nConclusions\n\n1. Early diagnosis is essential to prevent disease progression, complications, and improve the patient’s quality of life.\n\n2. Likewise, it is of high relevance to determine the form of presentation of the disease to achieve optimal response to treatment and a favorable prognosis. Similarly, the possibility of atypical cases whose age of presentation is not expected should be taken into account.\n\n3. Because there are currently no specific guidelines for the management of Morphea, we recommend that physicians should customize the treatment according to the clinical characteristics of each patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images were obtained from the patient.", "appendix": "References\n\nFlorez-Pollack S, Kunzler E, Jacobe HT: Morphea: Current concepts. Clin Dermatol. 2018; 36(4): 475–86. PubMed Abstract \| Publisher Full Text\n\nTeske NM, Jacobe HT: Using the Localized Scleroderma Cutaneous Assessment Tool (LoSCAT) to classify morphoea by severity and identify clinically significant change. Br J Dermatol. 2019. PubMed Abstract \| Publisher Full Text\n\nLaxer RM, Zulian F: Localized scleroderma. Curr Opin Rheumatol. 2006; 18(6): 606–13. PubMed Abstract \| Publisher Full Text\n\nAranegui B, Jiménez-Reyes J: Morphea in Childhood: An Update. Actas Dermosifiliogr. 2018; 109(4): 312–22. PubMed Abstract \| Publisher Full Text\n\nTeske N, Welser J, Jacobe H: Skin mapping for the classification of generalized morphea. J Am Acad Dermatol. 2018; 78(2): 351–7. PubMed Abstract \| Publisher Full Text\n\nAdrovic A, Şahin S, Barut K, et al.: Juvenile Scleroderma: A Referral Center Experience. Arch Rheumatol. 2018; 33(3): 344–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMertens JS, Seyger MMB, Thurlings RM, et al.: Morphea and Eosinophilic Fasciitis: An Update. Am J Clin Dermatol. 2017; 18(4): 491–512. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLu MC, Shyur SD, Lee LW, et al.: Unilateral generalized morphea: First case report in Taiwan. Asian Pac J Allergy Immunol. 2018. PubMed Abstract \| Publisher Full Text\n\nElloudi S, Baybay H, Gallouj S, et al.: [Localized scleroderma: about 24 cases]. Pan Afr Med J. 2018; 29: 53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDańczak-Pazdrowska A, Polańska A, Synakiewicz J, et al.: Morphea and antithyroid antibodies. Postepy Dermatol Alergol. 2018; 35(5): 470–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRongioletti F, Ferreli C, Atzori L, et al.: Scleroderma with an update about clinico-pathological correlation. G Ital Dermatol Venereol. 2018; 153(2): 208–15. PubMed Abstract \| Publisher Full Text\n\nHassani J, Feldman SR: Phototherapy in Scleroderma. Dermatol Ther (Heidelb). 2016; 6(4): 519–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMertens JS, Marsman D, van de Kerkhof PC, et al.: Use of Mycophenolate Mofetil in Patients with Severe Localized Scleroderma Resistant or Intolerant to Methotrexate. Acta Derm Venereol. 2016; 96(4): 510–3. PubMed Abstract \| Publisher Full Text\n\nCareta MF, Romiti R: Localized scleroderma: clinical spectrum and therapeutic update. An Bras Dermatol. 2015; 90(1): 62–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi SC, Pope E: Localized Scleroderma. In: Textbook of Pediatric Rheumatology. [Internet]. Elsevier. 2016; 406–417.e4. Publisher Full Text\n\nFett NM: Morphea (Localized Scleroderma). JAMA Dermatol. 2013; 149(9): 1124. PubMed Abstract \| Publisher Full Text\n\nWardhana, Datau EA: A patient with plaque type morphea mimicking systemic lupus erythematosus. Acta Medica Indones. 2015; 47(2): 146–52. PubMed Abstract\n\nWalker D, Susa JS, Currimbhoy S, et al.: Histopathological changes in morphea and their clinical correlates: Results from the Morphea in Adults and Children Cohort V. J Am Acad Dermatol. 2017; 76(6): 1124–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDharamsi JW, Victor S, Aguwa N, et al.: Morphea in adults and children cohort III: nested case-control study--the clinical significance of autoantibodies in morphea. JAMA Dermatol. 2013; 149(10): 1159–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDenton CP, Khanna D: Systemic sclerosis. Lancet. 2017; 390(10103): 1685–99. PubMed Abstract \| Publisher Full Text\n\nNarbutt J, Hołdrowicz A, Lesiak A: Morphea - selected local treatment methods and their effectiveness. Reumatologia. 2017; 55(6): 305–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSartori-Valinotti JC, Tollefson MM, Reed AM: Updates on Morphea: Role of Vascular Injury and Advances in Treatment. Autoimmune Dis. 2013; 2013: 467808. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeibel L, Sampaio MC, Visentin MT, et al.: Evaluation of methotrexate and corticosteroids for the treatment of localized scleroderma (morphoea) in children. Br J Dermatol. 2006; 155(5): 1013–20. PubMed Abstract \| Publisher Full Text\n\nKnobler R, Moinzadeh P, Hunzelmann N, et al.: European Dermatology Forum S1-guideline on the diagnosis and treatment of sclerosing diseases of the skin, Part 1: localized scleroderma, systemic sclerosis and overlap syndromes. J Eur Acad Dermatol Venereol. 2017; 31(9): 1401–24. PubMed Abstract \| Publisher Full Text\n\nUziel Y, Feldman BM, Krafchik BR, et al.: Methotrexate and corticosteroid therapy for pediatric localized scleroderma. J Pediatr. 2000; 136(1): 91–5. PubMed Abstract \| Publisher Full Text" }	[ { "id": "69271", "date": "24 Aug 2020", "name": "Tasleem Arif", "expertise": [ "Reviewer Expertise Morphea", "Systemic sclerosis", "Linear atrophoderma of Moulin", "Erythromelanosis follicularis facei et colli", "idiopathic atrophoderma of pasini and Pierini", "Nanotechnology in dermatology and cosmetics" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have tried to present a case of generalized morphea in a postmenopausal woman in which they showed how a treating physician can face therapeutic challenge while treating a case of generalized morphea. However, there are major issues with this manuscript.\nAuthors have labelled it as isomorphic morphea which is not correct. Isomorphic morphea (after the isomorphic phenomenon of Koebener) occurs at the sites of repetitive trauma like bra line, waistband, inguinal creases, etc. As per authors patient had lesions on arm, and lower extremities which can’t explain the isomorphic phenomenon.\n\nThe examination part is very deficient. “Painless skin lesions without erythema “Authors should mention the type of lesion like macule, papule or plaque rather than using word skin lesion. Authors have not given detailed examination about the plaques present on lower extremities and what was the size of plaques. The broad term like lower extremities should be avoided. Authors have to actually describe the location of plaques, feet, leg or thighs.\n\nThe language used to describe the examination is not precise. Instead using words like “presenting plaques with indurated hiccups and hyperpigmentation” authors can simply say “hyperpigmented indurated plaques”.\n\nAuthors need to mention what was in the report of biopsy rather than simply mentioning that reported morphea. As there are depigmented areas in the plaques, Lichen sclerosus et atrophicus provides a good differential for such lesions.\n\nImages are of poor quality especially Fig 3. Authors have used “Inactive lesion in left forearm, Inactive lesion at the level of the right iliac crest, etc for illustrating figures which is not correct. Just visualizing a plaque you can’t state that it is inactive. There can be subclinical activity going on inside the plaque. It must be corrected.\n\nHow was phototherapy given? Authors have not mentioned which protocol was used, based on skin phototype or what. The dose of UVA 3.10–3.94 eV looks strange as the usual doses of UV light are in mj or joules. The dose needs to be standard one. Sun protection and sun blocks have not been recommended after phototherapy for the patient. Since patient had disseminated plaques, was any special advice given to protect the face of patient during phototherapy.\n\nWhen patient received phototherapy 5 times a week she didn’t get the benefit but once it was used twice a week patient got benefit. Can authors throw light on this phenomenon?\n\nHow was the improvement of lesions assessed? Authors have not used any objective tool (like durometer, cutometer, USG, The Localized Scleroderma Cutaneous Assessment Tool) to assess the change in the lesions. Even the authors have not commented on the induration or pigmentation or size reduction of lesions. Then how was the improvement measured?\n\nMethotrexate was administrated orally (7.5 mg twice a week). Was folate given along with it? Which regimen was followed by authors in giving methotrexate twice a week? Was once weekly safer option and might not have impaired patients liver profile. I feel authors have been very superficial in most of the clinical part of the case.\n\nFor diagnosing generalized morphea authors must have stated the actual number of lesions and their size as per the classification system which they have mentioned (Laxer and Zulian).\n\nThe manuscript has several language and grammatical errors and needs to be written by an expert medical writer. There are incomplete sentences with many having punctuation mark mistakes.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] }, { "id": "88037", "date": "20 Jul 2021", "name": "Takamitsu Makino", "expertise": [ "Reviewer Expertise Scleroderma" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report shows that the phototherapy using UVA is effective for a patient with generalized morphea.\nPlease show the clinical photography of the active skin lesions and time-course of phototherapy.\n\nPlease show the clinical examination for the diagnosis, such as anti single-stranded DNA antibody, anti-histone antibody, rheumatoid factor, IgG and soluble IL-2 receptor. The authors should distinguish from the Borrelia infection.\n\nThe authors should discuss why the second phototherapy was more effective for skin lesions.\n\nIs the background of the case’s history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] } ]	1	https://f1000research.com/articles/9-107
https://f1000research.com/articles/9-106/v1	11 Feb 20	{ "type": "Research Article", "title": "4K ultra HD technology reduces operative time and intraoperative blood loss in colorectal laparoscopic surgery", "authors": [ "Giulio M. Mari", "Jacopo Crippa", "Pietro Achilli", "Angelo Miranda", "Letizia Santurro", "Valentina Riggio", "Martino Gerosa", "Pietro Ascheri", "Giuseppe Cordaro", "Andrea T.M. Costanzi", "Dario Maggioni", "Giulio M. Mari", "Jacopo Crippa", "Angelo Miranda", "Letizia Santurro", "Valentina Riggio", "Martino Gerosa", "Pietro Ascheri", "Giuseppe Cordaro", "Andrea T.M. Costanzi", "Dario Maggioni" ], "abstract": "Background: HD systems are routinely used in laparoscopic surgery, 4K ultra HD monitors are mainly available within specialized, high-volume laparoscopic centers. The higher resolution of 4K ultra HD video could upgrade the surgical performance improving intraoperative and post-operative outcomes. Methods: We performed a retrospective comparative analysis of intraoperative parameters and post-operative outcomes in a cohort of patients operated on for elective laparoscopic procedures for colo-rectal cancer during two different time frames: 2017 procedures performed using the Visera Elite full HD technology (® Olympus America, Medical) and the 2018 procedures performed the Visera 4K Ultra HD System (® Olympus America, Medical). Results: There was a statistically significant reduction in operative time in patients operated on with the 4K ultra HD technology compared to HD technology (p < 0.05). Intraoperative blood loss was significantly reduced in patients operated in 2018 (p < 0.05). There were no statistically significant differences in complication rate and postoperative outcomes between the two groups.", "keywords": [ "Colorectal surgery", "laparoscopy", "4K full HD" ], "content": "Introduction\n\nLaparoscopic surgery provides several intra- and post-operative advantages such as improved cosmesis, less post-operative pain and faster recovery compared to open surgery1. The development of new technologies has allowed laparoscopic surgery to become faster and safer. In particular, the quality of digital imaging has played a key role in the development of such procedures2. The association between quality of vision and surgical outcome is very tight. Improving the image quality allows the sharpness of laparoscopic surgery to be fully expressed. The clear view of the anatomical landmarks can improve the accuracy of dissection, permitting dissection of the lymphatic and nervous structures, which represent a crucial aspect of colorectal surgery3. Furthermore, the improved visualization of vascular structures and the better definition of adipose tissues may avoid intraoperative bleeding and reduce operative time, resulting in reduced surgical stress for the patient and potentially decreased complication rate4.\n\nRecently, several new imaging technologies, such as three-dimensional (3D)/high-definition (HD) stereovision and high-resolution two-dimensional (2D)/4K monitors have been made available to laparoscopic surgeons5. However, it is still unclear to what extent these technologies can actually improve surgical performance6.\n\nNowadays, HD systems are routinely used in laparoscopic surgery, while 4K ultra HD monitors are mainly available within specialized, high-volume laparoscopic centers7,8. The higher resolution of 4K ultra HD video could upgrade the surgical performance by improving depth perception, dissection precision, and bleeding control, but no study has yet provided a good evidence to sustain this finding. For this reason, we designed the present study to compare surgical intraoperative and post-operative parameters in patients operated on for colorectal cancer either with 4K ultra HD technology or a standard HD vision.\n\n\nMethods\n\nEthical approval was sought for the present study and the need for approval was waived by the ASST-MONZA ethics committee. According to the ASST-MONZA hospital regulation and to the local ethics committee, retrospective studies which do not involve a clinical intervention do not need to undergo the institutional review board ethical approval process. Written informed consent for reanalysis of the data and revision of clinical images and videos was taken from all the patients at the day of the inpatient visit one week before the surgical intervention date, as per routine practice in our institution9.\n\nWe performed a retrospective analysis of consecutive adult patients operated on for elective laparoscopic procedures for colorectal cancer during two different time frames: 2017 procedures performed using the Visera Elite full HD technology (® Olympus America, Medical) and the 2018 procedures performed the Visera 4K Ultra HD System (® Olympus America, Medical). Patients were divided into groups according to optic technology used. All the operations were performed in one hospital with the same operating room settings during the study period, except for the optic technology used. Patients were excluded from the analysis if they were submitted to surgery for benign disease, for palliative resections or in an emergency setting.\n\nAll procedures in 2017 and 2018 were performed with the same hybrid energy device. Surgical procedures were: laparoscopic right hemicolectomy (LRH) with intra-corporeal anastomosis; laparoscopic left hemicolectomy (LLH) with the anastomosis performed according to the Knight-Griffen technique; laparoscopic rectal anterior resection (LRAR) with the anastomosis performed according to the Knight-Griffen technique and loop ileostomy10. All procedures were performed by four colorectal laparoscopic surgeons, each of them with more than 100 totally laparoscopic procedures involving right colectomies, left colectomies and rectal anterior resections performed before 2017. Operating surgeons were asked to provide feedback on the ability of 4K ultra HD technology to visualize anatomical structures with respect to the HD technology, and regarding the eye fatigue perceived at the end of a procedure with 4K ultra HD technology over the HD technology. Feedback was collected through a written survey that was filled out at the end of each surgical procedure. This survey was routinely completed by the staff surgeons at the end of every minimally invasive procedure as part of the surgical intervention notetaking. The surveys were then retrieved form the electronic medical records.\n\nData were derived from the prospectively maintained electronic database of medical records of our institution. This database contained demographic information, admission dates and discharge diagnoses of each patient undergoing surgery for colorectal cancer. Electronic medical records contained details of every inpatient hospitalization at our institution, every outpatient visit to the clinic or emergency department, as well as every laboratory result and all data concerning the postoperative course after surgery. Specific items in the database are described in Table 2. Time of surgery, blood loss, conversion rate, as well as complication rate according to Clavien-Dindo (CD) classification, were analyzed11. We also focused on CD complication rate for major complications (CD major or equal to 3).\n\nDescriptive statistics for categorical variables were reported as frequencies (%), continuous variables as mean (standard deviation) or median (interquartile range (IQR)) according to distribution. The Chi-square2 test was used to compare categorical variables. All statistical tests were two-sided and a level of less than or equal to 0.05 was used to indicate statistical significance. Data analysis was performed with Statistical Software for the Social Sciences (SPSS) Advanced Statistics 22 (IBM Software Group, 200 W. Madison St., Chicago, IL; 60,606 USA).\n\n\nResults\n\nThere were no statistically significant differences in terms of baseline characteristics between groups9. Table 1 describes patient characteristics.\n\nBMI, body mass index; SD, standard deviation; ASA, American Society of Anesthesiologists’ physical status classification; LLH, laparoscopic left hemicolectomy; LRH, laparoscopic right hemicolectomy; LRAR, laparoscopic rectal anterior resection.\n\nIn 2017, a total of 28 LHRs, 38 LLHs and 28 LRARs were performed, while there were 30 LRHs, 44 LLHs and 23 LRARs in 2018. The overall complication rate according to CD classification was 21.2% (20/94) in 2017 and 17.5% (17/97) in 2018 (p>0.05). The rate of major complications (CD ≥ 3) was 6.3% (6/94) in 2017 vs 4.1% (4/97) in 2018 (p>0.05). In particular, there were no statistically significant differences in anastomotic leak rate, post-operative bleeding rate, mortality rate and readmission rate between groups. Mean surgical time was statistically significantly shorter with 4K ultra HD technology than HD technology (p < 0.05). Intraoperative blood loss was statistically significantly reduced in patients operated on in 2018 (p < 0.05) (Table 2).\n\nCD, Clavien-Dindo; P.O., post-operative; SD, standard deviation.\n\nFigure 1A shows the ileocolic vein and the inferior mesenteric vein dissected during a right hemicolectomy with complete mesocolic excision. Figure 1B shows the middle colic vein isolated during the same procedure performed with 4K ultra HD technology. Figure 1C shows the Gerota fascia and the inferior mesenteric artery isolated during a left hemicolectomy with HD technology. Figure 1D shows the hypogastric plexus at the origin of the inferior mesenteric artery spared during anterior rectal resection with 4K ultra HD technology.\n\nA) Ileocolic vein and inferior mesenteric vein (HD technology). B) Middle colic vein (4K ultra HD technology). C) Gerota’s fascia and inferior mesenteric artery (HD technology). D) Hypogastric plexus at the origin of the inferior mesenteric artery (4K ultra HD technology). Images were obtained retrospectively from the video electronic library of our institution, which contains records of all minimally invasive procedures of patients who gave consent for publication of the images for educational or research purposes.\n\n\nDiscussion\n\nThe main finding of our study is the statistically significant reduction in surgical time with the 4K ultra HD technology compared to normal HD technology. Intraoperative blood loss was also statistically significantly reduced in patients operated on with 4K ultra HD technology.\n\n4K ultra HD monitors were introduced in our surgical department in January 2018, replacing full HD technology for major colorectal laparoscopic procedures. Accurate visualization of the anatomical structures is crucial during laparoscopic procedures. Recognizing the vascular structures, their divisions and their course within the mesocolic fat tissue is of fundamental importance in oncological surgery12. Lymph node visualization along the course of the vessels and the ability to remove them without encountering troublesome bleeding allow a more precise dissection13.\n\nCutting edge technologies like 4K ultra HD allow the surgeon to be even more minimally invasive. Abderlahman et al. reported on the importance of the quality of 4K ultra HD images in reducing mistakes in structure identification1. Moreover, during left sided colorectal laparoscopic procedures, a precise visualization of the autonomic nerves is related to a better preservation of the genitourinary function. Especially for LRARs, both preservation of the autonomic nerves and avoiding injury to the mesorectum are essential for functional outcomes14.\n\nThe results we reported show how, following the introduction of a 4K technology, operating time for the same operations performed by the same surgeons decreased significantly. This may lead to lower operating room costs, as well as to the clinical benefits of a reduction in the peri-operative stress that patients are subjected to due to the duration of surgery and use of anesthesia15. The significant reduction of intraoperative blood loss in the two groups could be related to the improved capability in recognizing vascular structures that 4K ultra HD permits.\n\nAlthough it was not scientifically investigated in the present work, the subjective impressions of the operating surgeons are important when a new technology is introduced. All participating surgeons subjectively reported a much more detailed image with the 4K ultra HD technology. They all depicted a clearer understanding of the anatomical structures and of the surgical planes. In LRARs above all, the advantages of the 4K ultra HD view have been particularly enhanced, given the complexity of the anatomical field16,17. All operators reported ending procedures with less eye fatigue using 4K technology, although this could not be measured. The eye fatigue was reported to be minimal during the procedures performed in 2018. This finding is important when related to the fact that in almost all cases, the surgeon has to perform three or four procedures in the same day. Moreover, the 4K ultra HD technology could help in reducing the inevitable eye fatigue that aging brings18.\n\nThis study has several biases, beside its retrospective nature. The estimation of the validity of a technology cannot be entrusted only to the operative time required and instead consists of much more complex analysis. However, the reduction in surgical time is a reliable assessment of the technological improvement reached. The subjective reports of the intraoperative perception of the operating surgeons were not structured in intelligible questionnaires. Further efforts should be made to produce a specific tool to investigate the quality of the devices used in mini-invasive surgery.\n\n\nConclusions\n\n4K ultra HD technology applied to colorectal oncologic laparoscopic procedures is safe and effective in reducing surgical time and minimizing intraoperative blood loss compared to normal HD technology.\n\n\nData availability\n\nZenodo: 4K Ultra HD Technology Reduces Operative Time And Intra Operative Blood Loss In Colorectal Laparoscopic Surgery. https://doi.org/10.5281/zenodo.36033429\n\nThis project contains the following underlying data:\n\n- Patients characteristics and surgical outcomes.xls\n\n- Original surgical image files in JPG format\n\nZenodo: 4K Ultra HD Technology Reduces Operative Time And Intra Operative Blood Loss In Colorectal Laparoscopic Surgery. https://doi.org/10.5281/zenodo.36033429\n\nThis project contains the following extended data:\n\n- Informed consent.pdf (copy of consent form in Italian)\n\n- Informed consent (english version).pdf (copy of consent form in English)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nConsent\n\nWritten informed consent for publication of the patients’ data and clinical images was obtained from each patient involved.", "appendix": "References\n\nAbdelrahman M, Belramman A, Salem R, et al.: Acquiring basic and advanced laparoscopic skills in novices using two-dimensional (2D), three-dimensional (3D) and ultra-high definition (4K) vision systems: A randomized control study. Int J Surg. 2018; 53: 333–338. PubMed Abstract \| Publisher Full Text\n\nHarada H, Kanaji S, Hasegawa H, et al.: The effect on surgical skills of expert surgeons using 3D/HD and 2D/4K resolution monitors in laparoscopic phantom tasks. Surg Endosc. 2018; 32(10): 4228–4234. PubMed Abstract \| Publisher Full Text\n\nPai A, Melich G, Marecik SJ, et al.: Current status of robotic surgery for rectal cancer: A bird's eye view. J Minim Access Surg. 2015; 11(1): 29–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKinugasa Y, Sugihara K: Topology of the fascial structures in rectal surgery: Complete cancer resection and the importance of avoiding autonomic nerve injury. Semin Colon Rectal Surg. 2010; 21(2): 95–101. Publisher Full Text\n\nBuia A, Stockhausen F, Filmann N, et al.: 2D vs. 3D imaging in laparoscopic surgery-results of a prospective randomized trial. Langenbecks Arch Surg. 2017; 402(8): 1241–1253. PubMed Abstract \| Publisher Full Text\n\nSugawara M, Kanazawa M, Mitani K, et al.: SMPTE Periodical Ultrahigh-definition video system with 4000 scanning lines. SMPTE Motion Imaging J. 2003; 112(10–11): 339–346. Publisher Full Text\n\nYamashita H, Aoki H, Tanioka K, et al.: Ultra-high definition (8K UHD) endoscope: our first clinical success. Springerplus. 2016; 5(1): 1445. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOlympus: VISERA 4K UHD System. Reference Source\n\nMari G, Crippa J, Achilli P, et al.: 4K Ultra HD Technology Reduces Operative Time And Intra Operative Blood Loss In Colorectal Laparoscopic Surgery. 2019. http://www.doi.org/10.5281/zenodo.3603342\n\nGriffen FD, Knight CD Sr, Whitaker JM, et al.: The double stapling technique for low anterior resection. Results, modifications, and observations. Ann Surg. 1990; 211(6): 745–51; discussion 751–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDindo D, Demartines N, Clavien PA: Classification of surgical complications: a new proposal with evaluation in a cohort of 6336 patients and results of a survey. Ann Surg. 2004; 240(2): 205–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShen Z, Cheng J, Yin M, et al.: Evaluation of anatomical landmarks for transanal total mesorectal excision based on MRI. Asian J Surg. 2019; 42(6): 667–673. PubMed Abstract \| Publisher Full Text\n\nLee CHA, Wilkins S, Oliva K, et al.: Role of lymph node yield and lymph node ratio in predicting outcomes in non-metastatic colorectal cancer. BJS Open. 2019; 3(1): 95–105. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMari GM, Crippa J, Cocozza E, et al.: Low Ligation of Inferior Mesenteric Artery in Laparoscopic Anterior Resection for Rectal Cancer Reduces Genitourinary Dysfunction: Results From a Randomized Controlled Trial (HIGHLOW Trial). Ann Surg. 2019; 269(6): 1018–1024. PubMed Abstract \| Publisher Full Text\n\nGraziola E, Elena G, Gobbo M, et al.: [Stress, hemodynamic and immunological responses to inhaled and intravenous anesthetic techniques for video-assisted laparoscopic cholecystectomy]. Rev Esp Anestesiol Reanim. 2005; 52(4): 208–16. PubMed Abstract\n\nFerrara M, Kann BR: Urological Injuries during Colorectal Surgery. Clin Colon Rectal Surg. 2019; 32(3): 196–203. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpadaro S, Karbing DS, Mauri T, et al.: Effect of positive end-expiratory pressure on pulmonary shunt and dynamic compliance during abdominal surgery. Br J Anaesth. 2016; 116(6): 855–61. PubMed Abstract \| Publisher Full Text\n\nFergo C, Burcharth J, Pommergaard HC, et al.: Age is highly associated with stereo blindness among surgeons: a cross-sectional study. Surg Endosc. 2016; 30(11): 4889–4894. PubMed Abstract \| Publisher Full Text" }	[ { "id": "61891", "date": "29 Jul 2020", "name": "Matteo Uccelli", "expertise": [ "Reviewer Expertise Colorectal laparoscopic surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a retrospective analysis of a cohort of patients operated on for elective laparoscopic procedures for colo-rectal cancer during two different time frames. The main change reported among the two time periods is the introduction of a new visual technology. From this point of view, we can call this a “before and after trial”. The information provided is therefore not comparable with an eventual RCT since the introduction of new technology makes the old one un-applicable. Thus the data provided, although coming from a retrospective analysis can be reliable.\n\nThe surgical volume reported are adequate to consider the Unit where the study was performed a “high volume unit” solving the possible bias of the learning curve.\n\nThe main conclusion reached by the authors is that operative times could be reduced by the introduction of a 4K technology. Such a relationship between technology and surgical time is interesting although partial and it is commented by the authors in the limitation assuming that “the estimation of the validity of a technology cannot be entrusted only to the operative time required, but consists of a much more complex analysis. However, the reduction in surgical time is a reliable assessment of the technological improvement reached”. Also, the idea of structuring a tool to inquire about the surgeon’s perception when introducing a new technology is relevant and should be taken into consideration for future studies.\n\nReduction in intraoperative blood loss in the 4k patients is an important finding even if the incidence of post-operative bleeding is substantially equal in the two groups. From this point of view, the blood loss data correlates more with a precise dissection more than with the complication rate, scoring a point for the 4k technology. This trial is simple but clear. It describes the comparison in terms of surgical outcomes of two different visual laparoscopic technology in two similar time frames. It is an interesting attempt to evaluate the relationship between technical means and surgical activity which is oftentimes put aside. Statistical analysis are clear and well exposed. The background is fluid and contextualized. The discussion is clear and it does not assume more than what it should according to the nature of the trial itself. Limitations are reported and clearly understandable. As previously reported I think that this trial should be accepted for publication.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "59927", "date": "30 Jul 2020", "name": "Isacco Montroni", "expertise": [ "Reviewer Expertise Colorectal mini-invasive surgery." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe abstract is clear and well structured according to the guidelines of the journal. The introduction properly explains how technology and surgery have to get along improving the surgical outcomes. However, I think it is crucial that the results provided should be explained in a scientific way. From this point of view, this article is a quite good attempt towards this direction.\n\nLooking at the extended materials attached data are well collected and presented in a precise way. I believe that having the chance to see the original data is a great opportunity for whoever wants to read an article and therefore I congratulate with the journal and the authors.\n\nThe two cohorts of patients undergoing elective colo-rectal laparoscopic procedures are homogeneous in terms of tumor location, age, and sex and therefore are fully comparable. The authors are assuming that the introduction of new technology could have led to better surgical outcomes. The way they tried to prove it is a retrospective analysis that does not actually provide the best level of evidence. However, when you compare two different types of technologies, developing an RCT is really difficult and it could also be un-ethical.\n\nTaking a look at the results the authors depicted a reduction in blood loss and surgical time in favour of patients operated with the new technology. This result can be of some interest in its field even though cannot be considered conclusive of the comparison (as stated by the authors in the limitations). I think another important issue scoring a point for this trial is that it is not sponsored by the company providing the 4k technology. Usually, this kind of trial comes straight from the pharma. Here instead this bias can be solved by the fact that the authors conducted the study independently and therefore without having pressures from the providing company. We do not have to forget that the connection between clinical research and industry has lots of precious points but also lots of grey zones. The attempt done by the authors is from this point of view of great value and should be taken in serious consideration.\n\nFrom a purely practical point of view:\nThis study is well understandable.\n\nMethods are clear and complete.\n\nResults are well explained and as previously said, they come together with the full amount of data.\n\nStatistics is good enough.\n\nThe discussion could be implemented but still, there are not so many materials in the literature to compare these results with. This latter also shows how this simple study could be of some value/novelty.\n\nI consider the study suitable for indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-106
https://f1000research.com/articles/9-104/v1	11 Feb 20	{ "type": "Review", "title": "The challenges and mental health issues of academic trainees", "authors": [ "Renee Eleftheriades", "Clare Fiala", "Maria D. Pasic", "Renee Eleftheriades", "Clare Fiala" ], "abstract": "In the last decade, mental health issues have come to the foreground in academia. Literature surrounding student mental health continues to grow as universities try to implement wellness services and study the mental health of their students. Studies vary greatly in terms of measurement tools, timeframe, sample demographics, as well as the chosen threshold of symptom severity for diagnosis. This review attempts to summarize, contextualize and synthesize papers that pertain to the challenges faced by academic trainees at the undergraduate, graduate and post-graduate level. The evidence for, and against, the common claim of increasing prevalence of mental health issues among students in recent years is discussed. While some studies support this claim, it is difficult to reach a definitive conclusion due to numerous confounding factors such as increased help-seeking behaviour, greater awareness of mental health issues and weak methodology. The prevalence of depression, anxiety, suicidal and self-injurious behaviour, distress and general mental illness diagnoses are discussed. Other issues known to influence mental health, such as sexual assault and bullying, are briefly addressed. Finally, select studies on a few wellness strategies that may improve mental health of trainees, such as mindfulness, are summarised, along with diverse recommendations for individual students, universities, and academia as a whole.", "keywords": [ "academic training", "mental health", "trainee challenges", "anxiety", "depression", "graduate students", "academic jobs", "mindfulness" ], "content": "Introduction\n\nIn this review paper, we discuss the complex issues facing academic trainees and use many abbreviations summarized in Table 1 (references 1–14). Today, the pressure faced by university students at all levels may be greater than it has ever been in the past 25 years15. Increasing financial stressors since the 2007 recession are affecting undergraduates and graduates alike15–18. Students are increasingly concerned with getting a high-status job and earning a high income, and are consequently taking their academics more seriously15,19–21, resulting in increased feelings of academic pressure as early as high school15,19. This increased cultural focus on academia is reflected by the fast-paced growth in the number of PhD trainees, which is now outpacing the growth rate of the academic employment market22. As their numbers increase, graduate students face greater competition for a tightening pool of academic jobs, funding resources and mentorship16,23,24. These raised stakes underscore the pre-existing stresses and high competition inherent to academic culture25, resulting in intensely stressful conditions26. Thus, it is unsurprising that students have been repeatedly found to have worse overall mental health compared to the general population, characterized by elevated rates of depression, distress and other mental illnesses27–31.\n\nSeveral studies have posited that the prevalence and severity of university student psychological issues is on the rise according to counselling centres, self-reported mental health data and symptom severity scales3,32,33, though the validity of these findings has been debated34,35. Still, even though the exact trends in psychopathology are difficult to verify due to confounding factors, mental health is increasingly being recognized as an important issue. The Cooperative Institutional Research Program at Higher Education Research Institute (CIRP) of the University of California at Los Angeles found that, for incoming freshmen, “student self-rating of emotional health was at an all-time low and feeling overwhelmed in high school had been increasing” in 201115. In tandem with recent social movements to reduce stigma around mental illness and promote awareness, many universities are now trying to incorporate wellness into the student experience. This review will discuss some of the literature relevant to a variety of student mental health issues and some of the proposed solutions therein. Abbreviations used in this review can be found in Table 1.\n\n\nAre student mental health issues increasing in prevalence and complexity?\n\nThere is growing evidence suggesting that the prevalence and/or severity of university mental health issues has been increasing over the past half-century. Summarized in Table 2, this section will discuss some of this evidence, as well as its criticisms. Firstly, as discussed in the Introduction, the CIRP at the University of California, Los Angeles, asserted that freshman self-reported mental health is at an all-time low for the past 25 years, as a direct result of increasing economic pressures15. The study found that this deterioration in self-reported mental health coincides with an increase in student loan use and a decrease in financial independence due to decreasing job opportunities.\n\nPlease refer to Table 1 for abbreviations.\n\nTwenge et al.33 performed a cross-temporal meta-analysis of self-reported student mental health between 1938 and 2007. The authors chose to do a cross-temporal analysis to avoid the issue of response bias which results from people with depression not living as long as the average population. They also controlled for each generation’s propensity to lie and corrected their answers on psychopathology scales, adjusting for newer generations being more open to answering the survey honestly. Their assessment of mental health via the Minnesota Multiphasic Personality Inventory found that mental health worsened in each generation, including increased anxiety, depression, and dissatisfaction. These authors attribute this worsening to increased materialism and ambition. They comment that such ambition may be unrealistic, leading to eventual depression, and also that these goals may not be as fulfilling finding “meaning” in life and building interpersonal relationships.\n\nGallagher, in his National Survey of College Counselling Centers, reports that 94% of American college counselling centre directors agree that there is a continuously increasing number of students with severe psychological problems36. Almost 90% of directors reported an increase in anxiety disorders over the past 5 years, while 58% reported an increase in clinical depression. It is also worth noting that 69% “reported an increase in crises requiring immediate response”, such as a student with a suicidal ideation.\n\nIt is entirely possible that this increase in students seeking counselling is due to the reduction of social stigma around mental health. Hunt and Eisenberg35 explore this possibility and find that this increased prevalence in reported problems does coincide with increases in help-seeking behaviour, but that it is possible that mental illness severity is indeed increasing in tandem.\n\nBenton et al.3 performed an analysis of the severity and frequency of the problems presented to campus therapists between 1988 and 2001, using the Case Descriptor List, a report filed by the therapist at the end of the client’s case. They found a threefold increase in suicidal tendencies, a twofold increase in depression, and a fourfold increase in issues stemming from sexual assault, concluding that not only the prevalence, but also the severity, of student mental health problems were on the rise. Until 1994, relationship issues were the most common reason for seeking counselling, but thereafter, depression and anxiety took its place. However, they acknowledged that several studies using client self-report data or “objective” measurement scales (i.e. not dependent on the report of the client or psychologist) consistently find no such increase. This raises the possibility that, for unknown reasons, therapists perceive an increase in student issues that is not reflected by self-reports or other attempts based on objective measurements.\n\nIn fact, based on this possibility, there is a body of work disputing the claim that student mental health issues are increasing at all. Sharkin disputes the validity of using counselling practitioners’ perceptions as evidence for an increase in mental health issues37. He also offers a critique on the aforementioned Benton study, firstly on the basis of its methods of measuring distress, which he feels are too vague to produce conclusive results, and secondly on its potential sampling bias, since it only examined students already receiving counselling34. However, it is important to note that Benton et al. did find a likely-genuine increase in students seeking counselling for career-planning issues. The authors believe this increase to be objectively genuine (rather than an artefact of therapists’ perceptions) because the number of students seeking vocational counselling decreased upon the establishment of a career-development centre at the studied university, but then increased again later3. This increase in career concerns is consistent with that described by the CIRP15, Twenge et al.33, and Bishop19.\n\nCostello and colleagues found no difference in depression levels for young persons over a 30-year span in a review of 26 studies, although cohorts were not specific to university students38.\n\nStrepparava and colleagues performed a 5-year study of Italian students receiving counselling, but they did not find an increase in mental health issue severity or prevalence39. However, 5 years is a very short timeframe for assessing overarching trends in mental health issues37.\n\nCornish et al.40 studied university students over a 6–8 year span and found no significant increase in mental health problems, and Berger et al.41 and Franke et al.42, actually found a decrease in mental health issue severity over time. However, the latter two authors were both studying German students, and attribute the decrease to positive changes in academic conditions that are specific to Germany only (for example, massive reduction in tuition). Furr et al. also found that depression rates decreased from 81% to 53% between 1987 and 200143. Pledge et al. acknowledge a large body of works that have found increases in mental health issues up to the 1980s, but the authors themselves did not find any difference between 1989 and 1995. They hypothesize that mental health issues may have been increasing until the 1980s, but thereafter began to plateau44. Finally, in a systematic review of 24 studies, Ibrahim and colleagues found no trend of increasing prevalence of mental health problems between 1990 and 201027.\n\nThus, the trend in student mental health issue prevalence is a controversial topic. While the above studies may not be reflective of a true increase in mental health issue prevalence among students, they reflect the importance of well-staffed and well-resourced university counselling centres and reveal that mental health and wellness are increasingly becoming important to students and universities.\n\n\nDepression\n\nRates of depression reported among students vary greatly, depending on factors such as symptom scale, demographics and time period, as summarized in Table 3. This section will discuss various reports and contextualize their findings. The American College Health Association National College Health Assessment (ACHA NCHA) Fall 201845 reported that 17.3% of respondents (a mixed sample of about three quarters undergraduates and one quarter graduates) had accessed professional services for depression in the past 12 months.\n\nPlease refer to Table 1 for abbreviations.\n\nSystematic literature reviews have found massive ranges of depression incidence among students: Ibrahim et al. found a global range of 10-84.5% among undergraduates27 and Alfaris et al. found 8–70%46 among Saudi Arabian health science students specifically. These ranges include the extremes of the most well-to-do universities in rich regions, to highly competitive conditions in impoverished regions. The weighted mean global rate of depression found by Ibrahim et al. was 30.6% ,which is closer to the following studies discussed in this review (though the mean for non-medical students only was slightly higher, 35.6%).\n\nIndependent studies surveying student symptoms using the PHQ-9 (Patient Health Questionnaire 9) for depression reported 13.8%47, 17.3%17, 15.4%48, and 7.9%49 among American, and in the last case, Australian, students. A well-known 2007 study on Emory University undergraduate students found the highest depression rates among the PHQ-9 studies included in this paper; they reported that 29.6% of the respondents had mild depression, 30.6% had moderate depression and 6.6% had severe depression50. A study using the CES-D (Centre for Epidemiologic Studies depression) scale found 14.5% for the rate of students with severe depression and an additional 19% with mild depression symptoms, totalling to 33.5% of students having any form of depression51. Using the DASS-42 (Depression, Anxiety and Stress) scale, Bayram and Bilgel found a rate of 27.1% for depression among Turkish university students52. When Goldberg’s Anxiety and Depression scale was used in a Spanish study, a much higher rate was found—55.6%53. Boujut et al. report, in a review of French literature, depression rates of about 30%, with 6% of students suffering from a major depressive episode. In their own study on university freshmen, they reported that 18% scored as moderately-to-severely depressed using the BDI (Beck Depression Inventory)20.\n\nMany studies have also examined the prevalence of general feelings of depression or sadness among students, rather than depressive symptoms on a clinical scale. In a study on Australian first-year undergraduates, Hussain et al. found that 21.3% reported feeling “often/always unhappy”, and 9% often/always felt they had nothing to look forward to. However, this study also found that only 8% of first-year students had a diagnosis of either anxiety or depression54. It is worth noting that this study was performed in a rural area, so perhaps such settings have lower rates of mental health diagnoses for a number of possible reasons, including lower access to mental health services. Similarly, a UK study reported that 32% of undergraduate students felt “often or always” down/depressed in the past 4 weeks55. An American study found that 53% of students reported feelings of depression since having started college, but “depression” here was not given a standard definition by the authors but rather left up to the interpretation of the individual respondent. It is also worth noting that grades were the most common reason cited by students for their depressive feelings43.\n\n\nAnxiety\n\nThe ACHA NCHA Fall 2018 survey reports that 22.1% of a mixed student sample (about three quarters undergraduates and one quarter graduates) had been seen by a professional for diagnosed anxiety in the past 12 months45. In total, 62.3% reported having felt “overwhelming anxiety” in the past year, with 29.5% having felt that way in the past 2 weeks alone. The lowest rates of anxiety discussed in this paper were found by Zivin and colleagues, using the PHQ-9 (GAD-7) diagnosis scale: they reported rates of approximately 4%56, 9.8%17 and 5–7%48. Interestingly, an Australian study using GAD-7, the extended and updated version of the PHQ-9, found a significantly higher rate of 17.5%. The authors explain that this rate may be higher than that found by papers such as Zivin et al’s because the extended and thorough nature of the GAD-7 makes it a more sensitive tool for detecting anxiety than the PHQ-9. Since this rate is much closer to that of students found to have been professionally diagnosed with anxiety by the AHCA NCHA, it is likely that the GAD-7 can identify students that the PHQ-9 would miss49.\n\nHigher rates were found using other scales: two papers, one using the Goldberg’s Anxiety and Depression scale and one using the DASS-42 scale, both found rates of 47%52,53. Please see Table 4 for a summary of anxiety rates among students.\n\nPlease refer to Table 1 for abbreviations.\n\n\nOther mental health concerns\n\nSome literature does not specifically test for depression or anxiety but rather addresses the prevalence of general mental health concerns among subjects, as summarized in Table 5. In a selection of UK student surveys, 10–20% report having a mental health issue55,57–59. In a 2010 study of Australian undergraduates, 19.2% qualified as at-risk for a serious mental illness according to the K10 (Kessler) scale, and a further 64.7% show sub syndromic symptoms that classify them as at-risk of a mild/moderate mental illness. In a comparative age-matched sample of the general population, only 3% classified as at-risk for a serious mental illness, supporting the conclusion that students have significantly higher rates of mental distress compared to the general population31.\n\nPlease refer to Table 1 for abbreviations.\n\nUsing the PHQ-9 and SCOFF eating disorder scale, Zivin et al. measured student mental health at the beginning and end of a two-year period. They found that, at either given time point, 35% of students had some probable mental health issue (depression, suicidal thoughts, self-harm, anxiety, or an eating disorder)48. A very similar result was found by Bruffaerts et al., who measured freshman mental health with the WMH-ICS (World Mental Health International College Student) survey and found a prevalence rate of 34.9%60.\n\nMany other studies also examine the prevalence of elevated or clinically significant distress among students. A brief review by Marcotte and Lévesque reports a range of 21–31% among European literature25. Two additional studies included in the present review have found rates that are in-line with this range using the GHQ-12 questionnaire: 19% among Hungarian health students61, and 30% among Canadian undergraduates29. Much higher rates were reported by studies using measures other than the GHQ-12, except for one that used the DASS-42 scale and found a rate of 27%52. Bernaras and colleagues reported that 47.4–62.8% of students were above the clinical cut-off in various measures of distress using the CORE-OM (Clinical Outcomes in Routine Evaluation-Outcome Measure) scale5.\n\nRosenthal and Wilson reported an even higher prevalence of 83% for “moderate-to-severe” distress among undergraduates, using the Dysphoria Domain of the Trauma Symptom Inventory and Psychological Distress Scale, although only 9% of participants were classified as having clinically significant levels62. Finally, Hyun and colleagues reported that 44–45% of graduate students at University of California Berkeley self-reported a significant emotional or stress-related mental health concern63,64.\n\n\nSuicidality and self-Injury\n\nSuicidality can be classified into consideration, ideation, plans and attempt. Table 6 provides an overview of the discussed studies. Some studies that have made an effort to distinguish serious thoughts from ideation include Eisenberg et al.’s17 and Garlow et al.’s50, which reported 6.3% and about 10% of students had such thoughts within the year the study was performed. Furthermore, Garlow also found that 19.2% of the 10% group were currently suicidal at the time of the study. Overall, 16.5% of the entire student population reported either a suicide attempt or non-suicidal self-injury incident at any point in their lives. They found that 12% of participants had reported a suicidal ideation in the past 12 months45, 4.2% reported ideation currently often or always54, and 8.5% reported it since starting college43.\n\nPlease refer to Table 1 for abbreviations.\n\nAccording to the ACHA NCHA, 2% of undergraduates have attempted suicide in the past 12 months45. Boujut et al. reported a range of 9–15% for suicidal ideation and 3–10% for suicide attempts among students20. In total, 4.3% of French freshmen reported having suicide plans. Hussain and colleagues further reported that 3.7% of first years surveyed had thoughts of self-harm (often or always), and that the percentage of students who had attempted self-harm at some point in their lives were 17% female and 11% male54. For undergraduates, the rate of self-harm was 8.5% and the rate of suicide attempts was 2% in the past 12 months45. For pure-graduate groups, the non-suicidal self-injury (NSSI) rate was 7.3% in the past 4 weeks66.\n\n\nGraduate students and post-doctorate challenges\n\nGraduate students face additional academic pressures, having to juggle a myriad of responsibilities, such as attending courses, managing their projects and writing papers. Many graduate students feel unsure about their future career paths and are afraid that they will not find a permanent position in academia67. Surveys find that graduate students’ greatest concerns are their work-life balance, career security and success, financial security, and general uncertainty about their professional path68,69.\n\nAs mentioned in the introduction, academic positions and funding are becoming increasingly more competitive23,24. The Organization for Economic Co-operation and Development reported that in the last 20 years, the number of young PhDs has doubled and academic positions did not keep pace with this rapid growth22,70–73. A 2014 report by the National Science Foundation found that employment across the science and engineering fields was the lowest in the past 15 years. For fields outside of science and engineering, employment was at the lowest in the past 20 years73. An American study from 2014 estimated that, in engineering, there are only enough academic positions for 12.8% of PhD graduates71. Meanwhile, a Physics World article, using data from the Institute of Physics, estimated that just 1.7% of the UK postdocs in physics would go on to an academic position74.\n\nGraduate education is widely seen as a secure path to a high-status job, but in reality about a third of graduates do not even believe that their PhD significantly improves their job prospects68. Many are afraid that they will not be able to find a job at the end of their studies, and this stress has been found to contribute to depressive feelings among graduate students75.\n\nThis increasing competition is also affecting grant funding: a report in the magazine Nature found that funding has not been increasing in pace with the growing number of PhDs. Major grant providers, such as the NIH and the European Research Council, now award grants to less than 20% of applicants, most of whom will be older scientists with more established careers rather than young investigators.\n\nAmerican funding success rates have more than halved since 198067. A graduate student survey found that 50% of North American PhD trainees felt surprised by how difficult it was to secure funding67. The increased scarcity of funding and research resources is likely contributing to trainee mental health issues23,30.\n\nThis high competition is usually explained by academia’s emphasis on overwork. Especially in the sciences, academia has created a culture that venerates the idea of voluntary self-sacrifice for the collective advancement of humanity76,77. As a result, there is a persistent expectation on graduate students and young researchers in general, to overwork themselves24,30,78–82. Many academics “see suffering as a badge of honour”, as University of Derby Psychotherapist Gareth Hughes explains in an interview with Nature78. For many graduate students and other early-career researchers, it is not out of the ordinary to work 60-80 hours a week79,81,83, or at least be expected to84,85. One study on graduate students of economics, natural sciences and engineering found that about 55% work 41-60 hours per week, and approximately another 25% work 61-80 hours per week. According to this study, the majority of graduate students work far past the 9-5 hours expected in most jobs86. As Evans et al. put it “work-life balance is hard to attain in a culture where it is frowned upon to leave the laboratory before the sun goes down”30. Meanwhile, many graduate students struggle to make ends meet on their stipends alone, despite their long work hours69,87. The veneration of suffering in academia makes it difficult for graduate students to recognize their distress as a valid mental health issue rather than just the hardship expected in higher education88.\n\nConsequently, several studies report that graduate students experience elevated stress levels, even compared to undergraduate students. The Gradresources survey found that 37% of graduate students experienced “more stress than they can handle” as a direct result of their studies, with a total of 45% reporting more stress than they can handle in general. A 2015 University of Arizona study found that 73% of graduate students reported greater than average stress, with 23% describing their stress level as “tremendous”89. In one survey of PhD students, 62% reported worrying about their work even when they were not currently working86. The ACHA NCHA found that 61.4% of graduate students report greater than average stress, compared to 57% of undergraduates45.\n\nStudies comparing graduate student mental health to the general population’s found them to be at greater than two-to-six times the risk of depression and anxiety compared to the general population23,30,86. Specifically, Evans and colleagues found, using the PHQ-9 and GAD-7 scales, that graduate students experienced depression and anxiety at rates of 41% and 39%, respectively. Comparatively, they reported rates of 6% for the general population in other GAD-7 studies30. Of course, the comparison studies are chosen on the basis of using the same measurement scale as the primary study, even though they may have been performed under different conditions. For example, the Evans study has been criticized for using a German sample as comparison rather than an American survey84. Levecque and colleagues23 performed a thorough analysis of Flemish PhD student mental health as compared to control groups from other highly educated demographics (those in the general population, those who are employed, and those who are higher education students obtaining a degree other than PhD) using the 12-symptom scale General Health Questionnaire (GHQ-12). They found that PhD students were roughly 1.5-4 times at the risk of experiencing each of the 12 symptoms of anxiety/depression, compared to the three other groups. Further, 32% of PhD students reported four or more symptoms, which classifies them as being at serious risk of developing depression and other common mental illnesses.\n\nBarreira et al. reported that 18% of graduate students in economics, natural sciences and engineering scored at or above 10 on the PHQ-9 scale for exhibiting symptoms of moderate-to-severe depression and anxiety. This score corresponds to severity to such a degree that the symptoms would likely garner a formal diagnosis from a professional, according to the authors. They compare these rates to those for the age-matched general population, which were found in other studies to be 5.6% for depression and about 3.5% for anxiety86. Thus, graduate students were found to be 3–5 times more likely than the general population to experience moderate-to-severe depressive and anxious symptoms.\n\nA brief review90 of seven studies on graduate student mental health showed a range of 43-44.7% for experiencing emotional/stress-related problems, including symptoms of depression. The one exception to this rule was the Gloria & Steinhardt study91 which found a depression rate of only 29% but that 58% classified as “languishing”. One study featured in the review is a 2014 UC Berkeley study that employed the CES-D scale. It found that 47% of PhD students and 37% of Masters students scored within the clinical depression range75. These are similar to the results found by Evans et al., and not significantly higher than the percent of students at risk of mental illness found by Levecque et al. This study also corroborates Levecque et al.’s finding that higher education students outside of PhD programs, including Masters students, have slightly lower rates of mental illness relative to PhDs.\n\nAnother particularly informative study was conducted by Garcia-Williams, Moffit, and Kaslow. It found that more than 50% of the graduate sample reported feelings of anxiety and 95.4% reported “feeling nervous or worrying a lot” within the past 4 weeks. Also, within the past 4 weeks, 34.4% scored moderate-or-higher levels of depression on the PHQ-9, and 44.4% felt hopeless. Most strikingly, 7.3% reported suicidal ideation, 2.3% reported forming suicide plans, and 1.7% had committed NSSI within the past 4 weeks66. For comparison, the ACHA NCHA found an NSSI rate of 3.2% in the past 12 months among graduate students45. Information from this section is gathered in Table 7.\n\nPlease refer to Table 1 for abbreviations.\n\n\nDo graduate students struggle more than undergraduate students?\n\nDespite the extra responsibilities and pressures faced by graduate students, there is evidence that their mental health is slightly-to-significantly better than that of undergraduates66,92. For example, the ACHA NCHA found that graduates had slightly lower rates of diagnosed depression (14.8% vs 17.3% in undergraduates) and anxiety (19.5% vs 22.8% in undergraduates)45. Also, the ACHA NCHA graduate reference group showed lower rates of feelings of depression (32.9% having felt “too depressed to function” in past 12 months, 28.4% “experienced depression” in past 12 months ), anxiety (60.5% having felt “overwhelming anxiety” in past 12 months, 16.6% in past 2 weeks), NSSI (3.3%), suicide attempts (0.3% in past 12 months) compared to those in the undergraduate reference group (42.8% and 32.9% for depression, 63% and 31% for anxiety, 8.5% for NSSI and 2% for suicide attempt, all in respective order)45.\n\nIn contrast, the ACHA NCHA did find a slightly higher rate of suicidal ideation (past 12 months) among graduates (12.7% versus 12%), and other studies have also found that graduate students may have higher suicidality than undergraduates66. Meanwhile, Eisenberg and colleagues also found slightly lower rates of mental health issues among graduate students; with a prevalence rate of 11.3%, they were 2.5 percentage points less likely to suffer from depression than undergraduates, and almost 10% less likely to suffer from anxiety (3.8% versus 4.1%)56. In Gallagher and Taylor’s report, 80% of the 125 student suicides reported in 2014 were carried out by undergraduates93. Most conclusively, Wyatt and Oswalt directly compared undergraduate and graduate mental health and found that undergraduates are significantly worse-off in that regard92.\n\nThese effects may be a result of the contradicting forces of increased pressure and of selection bias: graduate students face more adult responsibilities and higher academic pressure. However, in order to get into graduate school in the first place, students must be able to meet a high standard of academic functioning. It is likely that the slightly lower rates of mental illness among graduates reported by some studies are due to such selection bias. Students with severe mental health issues are less likely to be able to continue to graduate school.\n\n\nMedical and health studies students\n\nIt has been suggested that medical students and other health science trainees have particularly high rates of mental distress25,46,84. For example, a Swedish study using the MDI (Major Depression Inventory) found that medical students have a depression rate of 12.9%76, more than 1.5x the prevalence they found in the general population (7.8%)94. As a result, there is an abundance of mental health studies focussing on these disciplines specifically. A review of global literature, performed by Puthran et al., concluded that 28% of medical students worldwide could be classified as clinically depressed95. Notably, this study did not find a significant difference in depression prevalence between medical and non-medical students. Meanwhile, a review on Saudi Arabian health science student mental health by Alfaris and colleagues found a weighted average of 47%, calculated from a range of results between 8–70%46. However, this result may not be representative of those in North America. For example, the authors explain that the lowest end of the range (8%) was from an American study, whereas the highest end (70%) was from a Pakistani study. Finally, the global review of literature by Ibrahim and colleagues actually found a lower prevalence of depression in medical students: 25.6% (calculated from a range of 10.3–59%), compared with 35.6% for non-medical students (calculated from a range of 10–84.5%)27. The authors note that this is contrary to expectation and suggest the explanation that medical students are perhaps better at recognizing their own mental health issues and reaching out for help. It is also possible that selection bias plays a role in this finding as well.\n\nTurning to American studies, a study of Illinois pharmacy school students found that, on the PHQ-9 scale, 31.9% registered as having mild depression, 16.9% as having moderate depression, 2.4% as having moderate-severe depression, and 1.2% as having severe depression. The study also noted that pharmacy students were twice as likely as the general population to report experiencing a depressive symptom79. Dyrbye et al. measured burnout among medical students and found a prevalence of 49.6–52.8% using the Maslach Burnout Inventory96,97. More strikingly, the prevalence they found for suicidal ideation was 11.2% over the past 12 months, though they calculated that, if all non-respondents were assumed to experience no ideation, the lowest possible prevalence was 5.8%. When respondents were asked instead if they had ever considered suicide at any point in their lives, the prevalence rose to just over a quarter. Wallin and Runeson, in another Swedish study, found even higher rates of suicidal ideation: 33% and 44% for students in their first and final years, respectively98.\n\nDyrbye and colleagues also compiled evidence for and against the claim that medical students experience elevated distress and rates of depression and anxiety compared to the general population. They conclude that the vast majority of studies with upper-year medical students found that they do indeed experience significantly poorer mental health than the general population28. This is summarized in Table 8.\n\nPlease refer to Table 1 for abbreviations.\n\n\nWho is most at risk?\n\nOut of the studies included in this review, those that measured gender differences almost unanimously found that women are at higher risk for certain mental illnesses, such as major depression and anxiety, distress, self-harm, and/or suicidal ideation. One such study of the different trends between male and female undergraduate mental illness, Conley and colleagues speculate that males are more likely to avoid dealing with their emotions (avoidant coping), whereas females are more likely to try to acknowledge their feelings and try to deal with them. While avoidant coping is generally not considered healthy, it has not been conclusively linked to greater psychopathology in males and it could potentially be a better strategy for dealing with the stresses of university. The study suggests that female students’ tendency to focus on their feelings in an attempt to remedy them may actually be exacerbating their distress99. Interestingly, this finding of increasing distress over time among females, but not males, was also found in a separate study on Canadian medical students. In this study, male students started the semester with elevated distress (one standard deviation above the general population average), whereas females started with average distress which increased to the elevated level (one standard deviation above the general population average) by the end of the semester100.\n\nThis higher propensity for distress may be somehow correlated with the finding that females have higher financial strain and insecurity. The Wisconsin-HOPE Lab found that females were 9 percentage points more food-insecure compared to males101. Stallman’s study found that females were slightly more likely to report “frequent financial stress” and “constant financial stress”, and less likely to report “no financial stress” as compared to males31. However, the author does not comment upon whether these differences are statistically significant. More conclusively, a UK study by the National Union of Students reported that females are 1.5 times more likely to worry frequently about finances, compared to males103.\n\nAdditionally, the Global Young Academy found that women had a slightly more pessimistic view of their career prospects, giving lower estimates of their chances to become a professor or land an academic or teaching position. Overall, women gave their career prospects an average rating of 0.1 lower than males, on a scale of 1-5. Also, women were more likely to report lack of support from superiors (50.6% versus 41.2%) and job rationalization (21.3% versus 13.1%), as well as gender discrimination (17.1% versus 4.6%), as obstacles to their career104. However, the authors warn that an existing sampling bias due to overrepresentation of women in the survey was not corrected, so these values should not be viewed as conclusive.\n\nWhile male students have lower psychological distress than female students, they comprise a much larger portion of student suicides35. For example, in his 2014 survey of university counselling centres across America, Gallagher reported that 70% of that year’s student suicide victims were male105. The UK Office for National Statistics reported that male higher-education students are almost three times as likely to commit suicide as females106. This is consistent with the general population: while females are more likely to have suicidal ideation and/or attempt suicide, males are much more likely to actually end their lives107. This could in part be due to differences in chosen method: males are twice as likely to use a firearm, whereas females are three times as likely to attempt to poison themselves107. Also, Bernaras and colleagues found that men were at a higher risk of doing “actual harm” to either themselves or others5.\n\nBesides sex differences in the preferred method of suicide, another reason for the higher suicide rate among males is that they are less likely to access mental health services or partake in wellness practices, perceiving a higher stigma around discussing personal problems and seeking professional help27,108. Gallagher’s 2010 National Survey of University Counselling Centres found that just 35% of student-counselling clients are male105. Bernaras and colleagues even cited this discomfort with disclosing personal issues as an explanation for the gender differences many studies have found in rates of psychological distress and related symptoms. Perhaps this gap is due in part to the fact that women are more likely to openly and honestly report their mental health issues and symptoms on surveys5. Also, Conley and colleagues reported that male students typically have less social support than females99, which may be another factor that discourages them from reaching out to seek help. Finally, studies on undergraduates report that females are significantly more prone to eating disorders54,99, whereas males are more prone to binge drinking54,109.\n\nStudies including a racial component also found that racial minority students are at higher risk of distress and other mental health issues. Smith et al. found that minority students exhibited worse mental health than white students, even at universities that had a high concentration of students from ethnic minorities109. Eisenberg and colleagues found that white students were less likely to screen positive for major depression on the PHQ-9, compared to any other race, with students identifying as “other race” having the highest likelihood17,47. They also found that students identifying as multi-racial were more likely to experience major depression, suicidal ideation, and NSSI17. This evidence points to the possibility that students who do not easily fall into a single socially defined racial category experience additional stress. Richardson et al. corroborated claims that minority students experience poorer mental health, but interestingly, they found white students to be at higher risk of alcohol dependence than black and Asian students110. Notably, Dyrbye et al.’s review of North American medical students revealed no link between ethnicity and mental health28. It is possible that medical students have a higher socioeconomic status overall and therefore medical students from an ethnic minority do not experience more economic challenges than white medical students. Students of colour, especially black students, are less likely to seek mental health services compared to white students62,111–113. Moreover, minority students may experience additional race-related stress, in the form of MEES (mundane extreme environmental stress, also called “microaggressions”), isolation, and stereotype threat12,111.\n\nAccording to the HOPE Lab, minority students were at a higher risk of food and housing insecurity compared to white students, with the exception of Asian students, who were at the lowest risk out of all the racial categories101. Native American students were at the highest risk for food insecurity (55%), housing insecurity (69%), and homelessness (19%), followed by black students (54%, 55%, and 13%, respectively), and “mixed/other” students (50%, 52%, and 17%). Hispanic students were at a slightly lower risk (47%, 51%, and 10%), followed by Middle Eastern/Arab students (43%, 49%, and 12%). White students had the second-lowest risks (37%, 42%, and 11%), followed by Asian students, who had the lowest risks across the board (36%, 37%, and 7%). Native American and “mixed/other” students experienced the highest prevalence of homelessness101. The elevated risk of homelessness for “mixed/other” students may correlate with their higher risk of psychological distress. In the same vein, the CIRP reports that in America, students at historically black colleges and universities report higher levels of financial insecurity, with one in five students unsure of whether they had the financial means to complete their degree15.\n\nIt is likely that the link between ethnicity and socioeconomic status plays some role in the increased prevalence of mental health issues among minority students. Socioeconomic status has been linked to worse mental health35,114,115, reduced access to mental health services, and reduced wellness behaviour35,108. Eisenberg and colleagues found that even growing up in a lower- socioeconomic-status household predicted worse mental health in students47. In fact, a NUS Insight survey reports that 36% of students report that they worry about finances so much that it affects their mental health103. A British study found that self-reported financial stress and debt were strongly correlated to worse overall mental health, including depression, anxiety, stress and alcohol abuse. Interestingly, though, this study did not find that family affluence could predict mental health issues, in contrast to the findings by Eisenberg et al.110. Cuéllar and Roberts, therefore, argue that studies exploring the link between racial identity and mental health should also control for the mediating effect of socioeconomic status. In their analysis of survey responses gathered from Latino students, they found that socioeconomic status plays a much more significant role in poor mental health compared to ethnic identity114.\n\nInterestingly, in a study of 1,773 undergraduates across four socioeconomic status groups, Rosenthal and Wilson found no disparities in mental health service utilization between sexes, socioeconomic status groups, or ethnicities62. The authors note that this is contradictory to the prevailing assumptions about service use disparity, but they stress that there are no identifiable errors or statistical anomalies that could have erroneously led to these results.\n\nAs may be expected, LGBTQ+ students face additional challenges, unique to their identity. There is evidence that homosexual students experience poorer mental health and higher suicidality compared to heterosexual students111,116 with bisexual students especially at risk. Kerr, Santurri & Peters found female bisexual students to be 3.1 times as likely to report a depression diagnosis, twice as likely to report anxious and depressive symptoms, and almost five times as likely to report suicidality and self-harm, compared to heterosexual female students. Lesbian students were twice as likely to report a depression diagnosis, five times as likely to report self-harm, and about four times more likely to report suicidality, compared to heterosexual female students. Bisexual women were overall significantly more likely to report depressive and anxious symptoms and suicidality compared to lesbian and heterosexual women116. Meanwhile, Liu and colleagues reported that bisexual students were 1.5 times more likely to report a mental health diagnosis or suicidality than homosexual students, and 3.9 times more likely compared to heterosexual students. Furthermore, they report that over half had experienced suicidal ideation and a quarter had made attempts on their lives111. Eisenberg and colleagues also found that bisexual students were at particular risk of experiencing depression47.\n\nAs before, this comes with a significantly increased risk of housing and food insecurity: homosexual students had greater risk than heterosexual students, and bisexual students had the highest risk of all. Bisexual students were about 10–15 percentage points more likely to report food and housing insecurity and homelessness compared to heterosexual students, and 5–7 percentage points more likely than homosexual students101.\n\nNot just students from sexual-orientation minority experience additional distress and needs insecurity, but also students from a gender-identity minority as well. A study of transgender and non- binary students found that 85% reported a mental health issue, with 25% explicitly relating to the challenges of being a gender-identity minority to their poor mental health. Many trans/non-binary students are reluctant to access mental health services due to the risk of landing a transphobic or ignorant counsellor. Slightly more than 25% of respondents described their previous counselling experiences as ambivalent or non-affirming towards trans/non-binary issues117. The obstacles facing gender minorities who need to access mental health services are particularly concerning in light of the fact that they are at much higher risk for mental illness30,111,117. Evans and colleagues found that transgender students were 12-21 percentage points more likely to report anxiety compared to cisgender males and females respectively, and 16-22 points more likely to report depression30. Liu and colleagues reported that transgender students were 1.9-2.4 times as likely to report mental health issues and suicidality compared to cisgender students. They have a much higher rate of distress compared even to bisexual students, with two-thirds experiencing suicidal ideation and one-third having made an attempt111. It is additionally concerning that some studies have reported an increase in the prevalence of transphobic bullying at universities118.\n\nA report on LGBTQ+ scientists in the physical sciences may be indicative of some of the factors at play in LGBTQ+ students’ mental health challenges: 18% of LGBTQ+ scientists, and in particular, 32% of non-binary scientists, reported being the target of discriminatory behaviour at their workplace119.\n\n\nWhat stops students from getting help?\n\nWhile great progress has recently been made in dismantling the stigma around mental health issues, stigma is likely still a primary obstacle standing between many students and the help they need119. Hussain et al. interviewed some of the first-year subjects in their study and concluded that stigma is still a concern among students who are considering seeking help. Students mentioned feeling that seeking help might be embarrassing, as they would appear unable to handle academic and social stresses. The authors further suggest that students in rural/small-town settings may feel that there is a higher social risk to seeking help, due to the reduced privacy/anonymity of such settings54.\n\nSimilarly, Eisenberg, Golberstein, and Gollust investigated the most common reasons that students who need help do not seek it. In total, 20% cited worrying about what others will think of them if they were in mental health treatment, 10% feared that it would somehow go onto their academic record and 9% feared that their parents would find out. Besides stigma, 32% reported simply lacking the time to seek help, and 8% lacked the finances. A total of 32% thought they would just get better by themselves over time, which may indicate a need for better awareness about the seriousness of mental health issues. It is also noteworthy that 51% said they felt that stress was just a regular part of school, though only 45% indicated that they felt they had no need for mental healthcare. A possible interpretation is that the 51% recognized they had more stress than they could handle, but thought it would be inappropriate to seek help for distressed caused by “normal/mundane” problems such as school113.\n\nInterestingly, Eisenberg and colleagues also found that students’ own internalized stigma of mental illness is a bigger obstacle to seeking help than their perception of public stigma. In comparing students’ own internalized stigma with their perception of public stigma, Eisenberg found large disparities, pointing to the possibilities that students either have an inflated sense of public stigma or are under-reporting their own internalized stigma120. The review by Storrie, Ahern, and Tuckett compiles evidence that the vast majority of students with emotional problems and/or severe distress are not receiving treatment and attributes stigma as a key reason121.\n\nIn Gallagher’s report, only 13% of people who had completed suicide had received counselling105, though this may be a case of selection bias: students in treatment would be less likely to resort to suicide. In another study on the persistence of student mental health problems, less than half of the students who had a persistent mental health problem over the two-year span of the study received help within that duration48. In Barreira et al.’s study of PhD students, only 27% of students with depression and 21% of students with anxiety were in treatment. Although this could simply reflect oversensitivity of the survey’s screening instruments, only 27% of students who self-reported suicidal thoughts were in treatment, which indicates that a majority of students with mood disorders are not receiving treatment86. Similar numbers were reported by Garlow et al., who found that 84% of students with suicidal ideation and 85% of students who screened as moderately-severely-to-severely depressed on the PHQ-9 were also not receiving treatment50. According to Hunt and Eisenberg35, similar rates of treatment among mentally ill students (less than 25%) were also reported by Blanco et al.122, though they also noted that this low rate of treatment is not significantly different from the rate among non-students. There is evidence suggesting that men experience greater stigma around mental illness and are therefore particularly unlikely to seek help123. This also seems to be case for Asian and Latino students120.\n\n\nFactors that could contribute to mental health concerns\n\nSexual victimisation has been positively linked to suicidal ideation, depressive feelings, psychological distress, and risk-taking behaviour in university student samples of men and women124,125. According to RAINN, American university students are in the age range with the highest risk for sexual assault, although female students are at a lower risk than female non-students126,127.\n\nEstimates of university sexual assault frequency vary greatly. For university students of all genders and degree levels, RAINN cites a sexual assault frequency of 11.2%126,127.\n\nFor female students, Stepakoff’s review found between 13-27% were survivors of sexual assault. Outlier studies estimate rates greater than 50%124. American Association of Universities (AAU) statistics cited by RAINN are firmly within this range, reporting a sexual assault frequency of 23.1% among female undergraduates. The reported frequency for female graduate/professional students is much lower, at 8.8%128. Furthermore, they report that only 1 in 5 female student victims report to law enforcement114, and only 1 in 6 receive assistance from victim services127,128.\n\nConversely, male students are much more likely to be sexually assaulted than their non-student counterparts. The AAU’s estimate is 5.4% of undergraduates and 2.2% for graduate/professional students have been sexually assaulted127,128. Turchik reports a range of 18.5%–31% for rates of unwanted sexual contact in the past academic year125. Furthermore, Turchik reports that the range becomes 34–58% when male students are asked to report incidences since age 16125. Men are less likely to disclose and seek help for their unwanted sexual experience, due in large part to myths about male rape128.\n\nThus, it is important to educate male students in particular about their right to report, and to dispel stigma around being a male sexual assault victim. Finally, gender minorities are at an elevated risk of sexual assault and rape, especially gender-queer/gender non-conforming/trans-identifying males127,128.\n\nA survey of graduate students across the fields of economics, engineering, and natural sciences found that 16% of graduate students have experienced sexual assault at some point during their PhD studies. Specifically, 21% of female students and 13% of male students reported such an experience. Overall, 62.5% of instances were perpetrated by peers whereas 19% were perpetrated by a professor86.\n\nIt is well-known that bullying negatively impacts mental health118. Bullying in academia can happen by peers or by authorities (supervisors, professors, mentors, etc.). A common form of supervisor-student bullying is academic exploitation, in which a supervisor takes credit for a student’s work129. In other cases, supervisor-student bullying can include making inappropriate, discriminatory, or derogatory comments130. In all of these cases, students can often feel as if they have no means of recourse, because they fear that their concerns will not be taken seriously by higher-ups, or that antagonizing their supervisor will impact their academic/professional careers129–131. Thus, it is difficult to comment on the frequency of these issues. The hierarchical nature of higher education makes it difficult for students to report their experiences, either because they are afraid of the consequences, or because they believe their experience is normal130–132.\n\nYamada et al. investigated bullying in a study of Canadian graduate students133. They reported that overall, 21% of students reported experiencing bullying by their supervisor, though very low percentages of students reported that the bullying was frequent. They found that the most frequent bullying types were “destabilization” (changing goals/responsibilities without informing the student, persistently undermining or demoralizing the student, etc.), which was experienced in some form by 40% of respondents, and “isolation” (ignoring the student, neglecting to give them important information, restricting their non-academic activities), which was experienced in some form by 37% of respondents, and finally, “overwork” (unreasonable pressure or deadlines), which was experienced by 32.9% of respondents. The four most common sub-behaviours were “undue pressure to produce work” (31.7%), “shifting goal posts without telling you” (28.8%), “adding or removal of areas responsibility without consultation” (24.5%), and “threat to personal standing” (26.6%). In terms of authorship issues, 41.3% reported that a supervisor or other faculty member received honorary authorship on their work133.\n\nFinally, in Frank et al.’s study of 16 medical schools, up to 63% reported having experienced belittlement by a professor and 71% experienced belittlement by a resident. The rates of students reporting harassment by these groups were 21% and 27%134. Issues between graduate students and supervisors are important to document, as Evans et al. found that a student’s relationship with their supervisor has significant impact on their mental health30.\n\nA well-studied form of bullying at the undergraduate level is cyberbullying. Kokkinos et al. mention that 1/3 of students reported involvement in bullying through online methods, such as over social media. Their literature analysis reported a rate of 9% to over 50% for reports of student victimization135. Myers and Cowie report that students in fraternities/sororities, women, and LGBTQ+ students are particularly vulnerable118.\n\nThe recently identified practice of “mobbing” is another type of bullying that academic culture may particularly lend itself to. Mobbing occurs when a team of colleagues collude to exclude, isolate, and demean a specific target. According to Seguin, universities are particularly unable to deal with this subtle form of bullying because they presume to be objective institutions that are “above” social issues such as bullying. In reality, their anti-harassment resources are unequipped to handle such situations. Issues often get passed off euphemistically as “personality clashes” and victims are left with no real recourse136.\n\nThe Wisconsin-HOPE Lab found striking amounts of needs-insecurity among American undergraduate students across 66 institutions. The authors report that 36% of students reported experiencing food and housing insecurity in the past month, and 9% were homeless in the past year. Needs insecurity was highest among community college students and marginalized groups101. The higher needs insecurity among marginalized groups is discussed in more detail in the “Who is most at risk?” section of this review.\n\nPerhaps unsurprisingly, needs insecurity was also higher among student-parents. In four-year programs, they were 9 percentage points more likely to be food-insecure and 6 percentage points more likely to be housing-insecure. In two-year programs, these differences were even more exaggerated101. In their studies of medical students, Dyrbye and colleagues found that student-parents are more likely to drop out97 and more likely to screen for depression on the CES-D scale, though debate exists as to whether this elevated depression risk applies to fathers and mothers, or mothers only28.\n\nIn the essay “Student-parents and higher education: a cross-national comparison”, Brooks explores the unique challenges of being a student-parent and recommends cultural attitude shifts and institutional supports that are necessary to properly accommodate them. The author cites “the temporal demands of being both a student and a parent of a young child; the paucity of on-site childcare facilities; restrictive ‘no child on campus’ policies; late availability of educational timetables; inconvenient timing of lectures and acute financial pressures” as a few of these challenges, as well as the conflict between their identities and expectations as students versus parents137.\n\nIn a Nature article, Powell interviewed an American mother who describes the challenges of being a PhD student and early-career scientist in a country with no formalized paid parental leave. This article describes a similar lack of on-campus childcare services and provisions, such as private lactation rooms. It also highlights restrictive conference policies that do not allow speakers or attendees to bring their infants with them138. Ultimately, both pieces point to the problematic expectation that academics must forgo family for career success, leaving student-and-scientist-parents with little support. According to Powell, childbirth is likely to coincide with the most crucial stage of a female early scientist’s career, forcing her to make difficult choices138. The GYA corroborates that scientist-mothers in particular point to work-life balance as an obstacle in their careers, significantly more than childless scientists or fathers104. Their recommendations are covered further in Table 9.\n\n\nWellness\n\nWellness is a broad concept that endorses a holistic view of health, wherein physical mental, and emotional well-being are equally important. It embraces self-care, stress-relief and management, healthy emotional coping, mindfulness, and reaching out for help when necessary. As such, it emphatically promotes mental-health awareness and stigma reduction, and, more broadly, cultivation of good mental/emotional framework for coping with stress and grievances in a healthy manner144. A key tenet of wellness is the value of prevention over treatment145. Prevention is achieved by prioritizing an individual’s well-being, being aware of one’s health needs and adapting one’s lifestyle accordingly.\n\nFocussing on staying healthy and being aware of signs of illness, whether physical or mental, is a more productive approach to health as opposed to waiting until after one is already sick. Physical wellness include exercising and eating well, emotional wellness can include building a social support group and implementing healthy coping strategies, and mental wellness can include mindfulness activities and counselling, if necessary. Wellness may have been considered unimportant, or even a distraction from work, in the past, but cultural attitudes are shifting, as evidence amasses showing that employee happiness is key to maximizing productivity146,147.\n\nSeveral studies have assessed the effectiveness of wellness practices such as meditation, mindfulness, counselling and mindful physical exercise. Conley, Durlak, and Dickson performed an analysis of 83 studies to assess the effectiveness of mental health promotion and prevention programs in higher education. Their review confirms the effectiveness of wellness programs in alleviating depressive and anxious symptoms and highlights cognitive behavioural therapy and mindfulness as the two most effective wellness strategies. The study further concluded that supervised, skill-oriented, class-format wellness programs are the most effective148. In their own experiment with a wellness seminar for first- year students, Conley and colleagues once again found that attendance and engagement with the wellness program are key to seeing improvements. Participants who regularly attended wellness seminars and practiced the skills they were taught showed better psychosocial and academic adjustment and better stress management149.\n\nIn another such review, Fernandez and colleagues examined the effectiveness of university mental health promotion and intervention programs, as well as the qualitative validity of the studies being analysed. The topic of the studies analysed varied greatly, so it is difficult to generalize their results here. However, their assessment of the program efficacy, combined with validity considerations, had an overall mixed tone. For example, a 1984 University of Illinois policy mandating students with suicidal ideation to receive four therapy sessions appeared to reduce suicides by 72%, but oddly, graduate/professional student suicides rose by 94.6% following this policy150. A Chinese study on the impacts of a “youth development” wellness course reported that it was effective in increasing positive and hopeful feelings, but Fernandez and colleagues deemed the study to be of questionable credibility due to its lack of detail151. A third study found that subjecting medical students to a stress-management lecture reduced their stress by 46.7%, but again, it was deemed of low-credibility due to its lack of control group. Also, many other studies included did not find that such programs had any significant positive impacts. One repeated finding among studies in the review that did seem conclusive, however, was the impact of grading systems on student health: grading systems with fewer increments seemed to reduce stress, depression, and anxiety151.\n\nUniversities are trying a variety of approaches to improve student mental health. For example, a few studies have explored the possibility of using tai-chi to foster wellness among students. One such study found no significant effects on mental health152, but another study found that tai chi chuan significantly decreased anxiety and improved sleep quality among young adults153. In fact, a systematic review of the impact of tai chi on higher-education student mental health concluded that it is an effective method of reducing anxious and depressive symptoms among students154. As mentioned earlier, mindfulness has been a hot topic in student wellness discussions. Vidic & Cherup reported that mindfulness relaxation was so effective among a sample of college students, that the test group went from higher stress than the control group to lower stress155.\n\nIn a review of empirical mindfulness studies, Keng, Smoski, & Robins analyse a growing body of credible evidence that general mindfulness practices have a positive impact of mental health156.\n\nHowever, the two university-sample studies in the review have conflicting findings: Bluth et al. found that mindfulness can uplift bad mood in students157, whereas Kuehner, Huffziger & Liebsch did not find any significant impact158,159. The authors explain that this may be due to differences in sample or in methodology156. Cook recommends the usage of cognitive behavioural therapy and wellness courses to alleviate student mental distress, based on evidence supporting their effectiveness119. Cook references Brown & Schiraldi, who found cognitive behavioural therapy to be significantly more effective in stress-reduction than a conventional stress-management course among students. Similarly, Kitzrow compiles evidence on the positive impact of counselling on both student mental health and academic performance32.\n\nCombining the wellness principles of mindfulness and exercise, Caldwell et al. studied the benefits of mindfulness established through movement-based courses for university students. They reported that that such courses were successful in reducing negative arousal and the prevalence of insomnia, but mood changes were only statistically significant through the mediating factor of mindfulness: students who showed an increase in mindfulness (via the Five Facet Mindfulness Questionnaire) also showed an increase in positive feelings, relaxation, self-regulatory efficacy, and sleep quality, and a decrease in perceived stress154. Finally, this paper also includes recommendations from papers and articles on policy and attitude changes that can hopefully make academia a more welcoming and less stressful environment. For convenience, these are presented in Table 9.\n\n\nConclusions\n\nAs shown in this review, there is a plethora of studies covering various aspects of student mental health, using diverse measurement tools and consequently, finding a very broad range of prevalence. Even among two studies using the same measurement tool, the reported prevalence of mental illness may differ significantly, depending on what level of symptom severity the authors choose as the cut-off.\n\nContinuing to refine and standardize measurement tools seems necessary. Given the numerous strains and concerns specific to students and trainees in academia, it would seem appropriate that studies should view student wellness within the context of these challenges and continue assessing student mental health in comparison to that of the general population. Such comparisons illustrate whether student-specific challenges contribute to poor mental health, and therefore direct the approach of student wellness initiatives. For example, student wellness initiatives can guide students to deal with student-specific challenges or recommend changes to mitigate these challenges at the institutional level, rather than simply directing students to mental health counselling. Of course, as indicated by even the lower end of the prevalence ranges in this review, ensuring that university mental health counselling is well-staffed, well-resourced and well-educated is paramount to student well-being. This review merely recommends that student mental health be viewed within the context of student-specific challenges and that institutions and institutionalized counselling focus on helping students to deal with these issues.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "References\n\nMorgan JF, Reid F, Lacey JH: The SCOFF questionnaire: a new screening tool for eating disorders. West J Med. 2000; 172(3): 164–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmerican Psychological Association: Beck Depression Inventory. Am Psychol Assoc. Reference Source\n\nBenton SA, Robertson JM, Tseng WC, et al.: Changes in counseling center client problems across 13 years. Prof Psychol. 2003; 34: 66–72. Publisher Full Text\n\nAmerican Psychological Association: Center for Epidemiological Studies- Depression. Am Psychol Assoc. Reference Source\n\nBernaras E, Cerretani P, Garay B: Prevalence and severity of psychological problems in university students. Brit J Guid Couns. 2018; 46(4): 418–428. Publisher Full Text\n\nPsychology Foundation of Australia: Depression anxiety stress scales (DASS). Psychol Found Aust. 2018. Reference Source\n\nPsychiatry & Behavioral Health Learning Network: Generalized Anxiety Disorder -7 (GAD-7). Psychiatry Behav Heal Learn Netw. Reference Source\n\nGAIN Coordinating Center: GAIN Instruments. Chestnut Heal Syst. Reference Source\n\nAssessment GL: General Health Questionnaire. GL Assess. Reference Source\n\nSolutions S: Kessler Psychological Distress Scale (K10). Stat Solut. 2014. Reference Source\n\nPsychTools: Major Depression Inventory (MDI). PsychTools. 2018. Reference Source\n\nBarnett TM, McFarland A, Miller J, et al.: Physical and Mental Health Experiences Among African American College Students. Soc Work Public Heal. 2019; 34(2): 145–57. PubMed Abstract \| Publisher Full Text\n\nFramingham J: Minnesota Multiphasic Personality Inventory (MMPI). PsychCentral. 2018. Reference Source\n\nAmerican Psychological Association: Patient Health Questionnaire (PHQ-9 & PHQ-2). Am Psychol Assoc. Reference Source\n\nPryor JH, DeAngelo L, Blake LP: The American freshman: national norms Fall 2011. Coop Institutional Res Progr High Educ Res Inst UCLA. 2011. Reference Source\n\nFullick M: My grief lies all within\"-PhD students, depression & attrition. Univ Aff. 2011. Reference Source\n\nEisenberg D, Hunt J, Speer N: Mental health in American colleges and universities: variation across student subgroups and across campuses. J Nerv Ment Dis. 2013; 201(1): 60–7. PubMed Abstract \| Publisher Full Text\n\nRudgard O: Universities have a suicide problem as students taking own lives overtakes general population. The Telegraph. 2018. Reference Source\n\nBishop J: The changing student culture: implications for counsellors and administrators. J Coll Stud Psychother. 1993; 6(3–4): 37–57. Publisher Full Text\n\nBoujut E, Koleck M, Bruchon-Schweitzer M, et al.: La santé mentale chez les étudiants : suivi d’une cohorte en première année d’université. Ann Med Psychol (Paris). 2009; 167(9): 662–8. Publisher Full Text\n\nCoughlan S: Rising number of stressed students seek help. BBC. 2015. Reference Source\n\nMaher B, Sureda Anfres M: Young scientists under pressure: what the data show. Nature. 2016; 538(7626): 444. PubMed Abstract \| Publisher Full Text\n\nLevecque K, Anseel F, De Beuckelaer A, et al.: Work organization and mental health problems in PhD students. Res Policy. 2017; 46(4): 868–79. Publisher Full Text\n\nWallis T, Kelley D: Trends in academia: the reality of tenure track. Insid Sch. 2018. Reference Source\n\nMarcotte J, Lévesque G: Anxiety and well-being among students in a psychoeducation program: The mediating role of identity. J Coll Stud Dev. 2018; 59(1): 90–104. Publisher Full Text\n\nEditorial: Misspent youth. Nature. 2016; 538: 427.\n\nIbrahim AK, Kelly SJ, Adams CE, et al.: A systematic review of studies of depression prevalence in university students. J Psychiat Res. 2013; 47(3): 391–400. PubMed Abstract \| Publisher Full Text\n\nDyrbye LN, Thomas MR, Shanafelt TD: Systematic review of depression, anxiety, and other indicators of psychological distress among U.S. and Canadian medical students. Acad Med. 2006; 81(4): 354–73. PubMed Abstract \| Publisher Full Text\n\nAdlaf EM, Gliksman L, Demers A, et al.: The prevalence of elevated psychological distress among Canadian undergraduates: findings from the 1998 Canadian Campus Survey. J Am Coll Health. 2001; 50(2): 67–72. PubMed Abstract \| Publisher Full Text\n\nEvans TM, Bira L, Gastelum JB, et al.: Evidence for a mental health crisis in graduate education. Nat Biotechnol. 2018; 36(3): 282–4. PubMed Abstract \| Publisher Full Text\n\nStallman HM: Psychological distress in university students: a comparison with general population data. Aust Psychol. 2010; 45(4): 249–57. Publisher Full Text\n\nKitzrow MA: The mental health needs of today’s college students: challenges and recommendations. NASPA J. 2009; 46(4): 646–60. Publisher Full Text\n\nTwenge JM, Gentile B, DeWall CN, et al.: Birth cohort increases in psychopathology among young Americans, 1938-2007: A cross-temporal meta-analysis of the MMPI. Clin Psych Rev. 2010; 30(2): 145–54. PubMed Abstract \| Publisher Full Text\n\nSharkin BS: Assessing changes in categories but not severity of counseling center clients’ problems across 13 years: Comment on Benton, Robertson, Tseng, Newton, and Benton (2003). Prof Psychol Res Pr. 2004; 35(3): 313–5. Publisher Full Text\n\nHunt J, Eisenberg D: Mental health problems and help-seeking behavior among college students. J Adolesc Health. 2010; 46(1): 3–10. PubMed Abstract \| Publisher Full Text\n\nGallagher RP: National survey of counselling center directors. J Coll Stud Psychother. 2010; 6: 37–57. Reference Source\n\nSharkin BS: Increasing severity of presenting problems in college counseling centers: a closer look. J Couns Dev. 1997; 75(4): 275–81. Publisher Full Text\n\nJane Costello E, Erkanli A, Angold A: Is there an epidemic of child or adolescent depression? J Child Psychol Psychiatry. 2006; 47(12): 1263–71. PubMed Abstract \| Publisher Full Text\n\nStrepparava MG, Bani M, Zorzi F, et al.: Does the severity of psychopathology of Italian students receiving counselling services increase over time? A 5-year analysis and a comparison with a clinical and non-clinical sample. Clin Psychol Psychother. 2017; 24(6): O1448–O1454. PubMed Abstract \| Publisher Full Text\n\nCornish J, Riva M, Henderson M, et al.: Perceived distress in university counseling center clients across a six-year period. J Coll Stud Dev. 2000; 41(1): 104–9. Reference Source\n\nBerger H, Granke G, Hofmann F, et al.: Mental health of students and its development between 1994 and 2012. Ment Heal Prev. 2015; 3: 48–56. Publisher Full Text\n\nFranke GH, Jagla M, Petrowski K, et al.: Psychological distress in students today and 20 years ago. Ment Heal Prev. 2017; 5: 1–4. Publisher Full Text\n\nFurr SR, McConnell GN, Westefeld JS, et al.: Suicide and depression among college students: A decade later. Prof Psychol Res Pr. 2001; 32(1): 97–100. Publisher Full Text\n\nPledge DS, Lapan RT, Heppner PP, et al.: Stability and severity of presenting problems at a university counseling center: a 6-year analysis. Prof Psychol Res Pr. 1998; 29(4): 386–389. Publisher Full Text\n\nAmerican College Health Association: American College Health Association-National College Health Assessment II: Canadian Reference Group Executive Summary Fall 2018. Am Coll Heal Assoc. 2018. Reference Source\n\nAlfaris E, Irfan F, Qureshi R, et al.: Health professions’ students have an alarming prevalence of depressive symptoms: exploration of the associated factors. BMC Med Educ. 2016; 16(1): 279. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEisenberg D, Gollust SE, Golberstein E, et al.: Prevalence and correlates of depression, anxiety, and suicidality among university students. Am J Orthopsychiatry. 2007; 77(4): 534–42. PubMed Abstract \| Publisher Full Text\n\nZivin K, Eisenberg D, Gollust SE, et al.: Persistence of mental health problems and needs in a college student population. J Affect Disord. 2009; 117(3): 180–5. PubMed Abstract \| Publisher Full Text\n\nFarrer LM, Gulliver A, Bennett K, et al.: Demographic and psychosocial predictors of major depression and generalised anxiety disorder in Australian university students. BMC Psychiatry. 2016; 16: 241. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarlow SJ, Rosenberg J, Moore JD, et al.: Depression, desperation, and suicidal ideation in college students: results from the American Foundation for Suicide Prevention College Screening project at Emory University. Depress Anxiety. 2008; 25(6): 482–8. PubMed Abstract \| Publisher Full Text\n\nGress-Smith JL, Roubinov DS, Andreotti C, et al.: Prevalence, severity and risk factors for depressive symptoms and insomnia in college undergraduates. Stress Heal. 2015; 31(1): 63–70. PubMed Abstract \| Publisher Full Text\n\nBayram N, Bilgel N: The prevalence and socio-demographic correlations of depression, anxiety and stress among a group of university students. Soc Psychiatry Psychiatr Epidemiol. 2008; 43(8): 667–72. PubMed Abstract \| Publisher Full Text\n\nGalindo SB, Moreno IM, Munoz JG: Prevalencia de ansiedad y depresión en una población de estudiantes universitarios: factores académicos y sociofamiliares asociados. Clin y Salud. 2009; 20(2): 177–187. Reference Source\n\nHussain R, Guppy M, Robertson S, et al.: Physical and mental health perspectives of first year undergraduate rural university students. BMC Public Health. 2013; 13: 848. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNeale I, Piggott L, Hansom J, et al.: Student resilience: unite students insight report. Unite Students. 2016. Reference Source\n\nZiven K, Eisenberg D, Gollust SE, et al.: Prevalence of mental health problems and needs in a college population. J Affect Disord. 2009; 117(3): 180–5. PubMed Abstract \| Publisher Full Text\n\nPereira S, Reay K, Bottell J, et al.: University Student Mental Health Survey 2018. Insight Netw Dig-in. 2018. Reference Source\n\nKerr H: Mental distress survey overview. NUS RUS. 2013. Reference Source\n\nBrown P: The invisible problem? Improving students’ mental health. High Educ Policy Inst Report 88. 2016. Reference Source\n\nBruffaerts R, Mortier P, Kiekens G, et al.: Mental health problems in college freshmen: Prevalence and academic functioning. J Affect Disord. 2018; 225: 97–103. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBírá É, Ádány R, Kása K: Mental health and behaviour of students of public health and their correlation with social support: a cross-sectional study. BMC Public Health. 2011; 11: 871. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRosenthal B, Wilson WC: Mental health services: use and disparity among diverse college students. J Am Coll Health. 2008; 57(1): 61–8. PubMed Abstract \| Publisher Full Text\n\nHyun J, Quinn B, Madon T, et al.: Mental health need, awareness, and use of counselling services among international graduate students. J Am Coll Health. 2007; 56(2): 109–18. PubMed Abstract \| Publisher Full Text\n\nHyun JK, Quinn BC, Madon T, et al.: Graduate student mental health: Needs assessment and utilization of counseling services. J Coll Stud Dev. 2006; 47(3): 247–266. Publisher Full Text\n\nEisenberg D, Hunt J, Speer N: Mental Health in American Colleges and Universities: variation across student subgroups and across campuses. J Nerv Ment Dis. 2013; 201(1): 60–7. PubMed Abstract \| Publisher Full Text\n\nGarcia-Williams AG, Moffitt L, Kaslow NJ: Mental health and suicidal behavior among graduate students. Acad Psychiatr. 2014; 38(5): 554–60. PubMed Abstract \| Publisher Full Text\n\nWoolston C: Graduate survey: Uncertain futures. Nature. 2015; 526(7574): 597–600. PubMed Abstract \| Publisher Full Text\n\nWoolston C: Graduate survey: A love–hurt relationship. Nature. 2017; 550: 549–552. Publisher Full Text\n\nConcerns THE: Stress and Relief for American Graduate Students: Results from a Nationwide Survey. Grad Resources. 2011. Reference Source\n\nCharbonneau L: Producing more PhDs. Univ. Affiars. 2010. Reference Source\n\nLarson RC, Ghaffarzadegan N, Xue Y: Too Many PhD Graduates or Too Few Academic Job Openings: The Basic Reproductive Number R0 in Academia. Syst Res Behav Sci. 2014; 31(6): 745–750. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcKenna L: The Ever Tightening Job Market for PhDs. The Atlantic. 2016. Reference Source\n\nNational Science Foundation: Doctorate recipients from U.S. Universities. Natl Sci Found. 2015. Reference Source\n\nSutton A: Academic aspirations. Phys. World. 2015; 18. Reference Source\n\nPanger G, Tryon J, Smith A: The Graduate Assembly Graduate Student Happiness & Well-Being Report 2014. Grad Assem. UC Berkeley. 2014. Reference Source\n\nHerzig RM: Suffering For Science: Reason and Sacrifice in Modern America. New Brunswick: Rutgers University Press; 2005. Publisher Full Text\n\nDiamandis EP: A Key to Success Is the Key that Opens and Closes the Lab Door. J Appl Lab Med. 2019; 4(1): 135–136. PubMed Abstract \| Publisher Full Text\n\nWoolston C: Mental health in academia is topic of the week at a sold-out UK meeting. Nature. 2019. Publisher Full Text\n\nWoo E: “A toxic culture of overwork”: inside the graduate student health crisis. Stanford Dly. 2019. Reference Source\n\nKim Y, Faber E: What medicine can teach academia about preventing burnout. Nat Careers. 2019. Reference Source\n\nWoolston C: Workplace habits: Full-time is full enough. Nature. 2017; 546: 175–177. Publisher Full Text\n\nPowell K: Young, talented and fed-up: scientists tell their stories. Nature. 2016; 538(7626): 446–449. PubMed Abstract \| Publisher Full Text\n\nWong P: How to humanize higher education and reduce human suffering. Int Netw Pers Mean. page President’s Column. 2006. Reference Source\n\nDuffy M, Thanhouser C, Derry H: A lack of evidence for six times more anxiety and depression in US graduate students than in the general population. Nat Biotechnol. 2019; 37(7): 711–712. PubMed Abstract \| Publisher Full Text\n\nDiamandis EP: Getting noticed is half the battle. Science. 2015; 349(6244): 206. PubMed Abstract \| Publisher Full Text\n\nBarreira P, Basilico M, Bolotnyy V: Graduate student mental health: lessons from American economics departments. Harvard Univ. 2018. Reference Source\n\nCatcheside K: Removing the barriers to postgraduate education. Guard. 2011. Reference Source\n\nAguisanda F: At the end of the road, a new start. Science. 2018; 362(6418): 1078. PubMed Abstract \| Publisher Full Text\n\nSmith E, Brooks Z: Graduate student mental health 2015. Natl Assoc Grad Students. 2015. Reference Source\n\nTsai JW, Muindi F: Towards sustaining a culture of mental health and wellness for trainees in the biosciences. Nat Biotechnol. 2016; 34(3): 353–5. PubMed Abstract \| Publisher Full Text\n\nGloria CT, Steinhardt MA: Flourishing, languishing, and depressed postdoctoral fellows: Differences in stress, anxiety, and depressive symptoms. J Postdoc Aff. 2013; 3(1): 1–9. Reference Source\n\nWyatt TW, Oswalt SB: Comparing mental health issues among undergraduate and graduate students. Am J Heal Educ. 2013; 44(2): 96–107. Publisher Full Text\n\nGallagher RP, Taylor R: National Survey of College Counselling. Am Coll Couns Assoc. 2014. Reference Source\n\nDahlin M, Joneborg N, Runeson B: Stress and depression among medical students: a cross-sectional study. Med Educ. 2005; 39(6): 594–604. PubMed Abstract \| Publisher Full Text\n\nPuthran R, Zhang MW, Tam WW, et al.: Prevalence of depression amongst medical students: a meta-analysis. Med Educ. 2016; 50(4): 456–68. PubMed Abstract \| Publisher Full Text\n\nDyrbye LN, Thomas MR, Massie FS, et al.: Burnout and suicidal ideation among U.S. medical students. Ann Intern Med. 2008; 149(5): 334–41. PubMed Abstract \| Publisher Full Text\n\nDyrbye LN, Massie FS Jr, Eacker A, et al.: Relationship between burnout and professional conduct and attitudes among US medical students. JAMA. 2010; 304(11): 1173–80. PubMed Abstract \| Publisher Full Text\n\nWallin U, Runeson B: Attitudes towards suicide and suicidal patients among medical students. Eur Psychiatry. 2003; 18(7): 329–33. PubMed Abstract \| Publisher Full Text\n\nConley CS, Kirsch AC, Dickson DA, et al.: Negotiating the tansition to college: developmental trajectories and gender differences in psychological functioning, cognitive-affective strategies, and social well-being. Emerg Adulthood. 2014; 2(3): 195–210. Publisher Full Text\n\nVitaliano PP, Maiuro RD, Russo J, et al.: Medical student distress. A longitudinal study. J Nerv Ment Dis. 1989; 177(2): 70–6. PubMed Abstract \| Publisher Full Text\n\nGoldrick-Rab S, Richardson J, Schneider J, et al.: Still hungry and homeless in college. Wisconsin HOPE Lab. 2018. Reference Source\n\nHunt K, Gable KN: Prevalence of depressive symptoms and obsessive-compulsive personality traits among pharmacy students. Curr Pharm Teach Learn. 2013; 5(6): 541–5. Publisher Full Text\n\nFinance F: NUS insight: financial woes affecting mental health. Futur Financ. 2016. Reference Source\n\nFriesenhahn I, Beaudry C: The global state of young scientists-project report and recommendations. Glob Young Acad. 2015. Reference Source\n\nGallagher R: National Survey of College Counseling Centers. Int Assoc Couns Serv. 2014. Reference Source\n\nCaul S: Estimating suicide among higher education students, England and Wales: experimental statistics. Off Natl Stat. 2018. Reference Source\n\nNational Institute of Mental Health: Suicide. NIH. 2017. Reference Source\n\nLyssenko L, Müller G, Kleindienst N, et al.: Life Balance - a mindfulness-based mental health promotion program: conceptualization, implementation, compliance and user satisfaction in a field setting. BMC Public Health. 2015; 15: 740. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith KM, Chesin MS, Jeglic EL: Minority college student mental health: Does majority status matter? implications for college counseling services. J Multicult Couns Devel. 2014; 42(2): 77–92. Publisher Full Text\n\nRichardson T, Elliott P, Roberts R, et al.: A Longitudinal Study of Financial Difficulties and Mental Health in a National Sample of British Undergraduate Students. Community Ment Health J. 2017; 53(3): 344–52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu CH, Stevens C, Wong SHM, et al.: The prevalence and predictors of mental health diagnoses and suicide among U.S. college students: Implications for addressing disparities in service use. Depress Anxiety. 2019; 36(1): 8–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarland AF, Lau AS, Yeh M, et al.: Racial and ethnic differences in utilization of mental health services among high-risk youths. Am J Psychiatry. 2005; 162(7): 1336–43. PubMed Abstract \| Publisher Full Text\n\nEisenberg D, Golberstein E, Gollust SE: Help-seeking and access to mental health care in a university student population. Med Care. 2007; 45(7): 594–601. PubMed Abstract \| Publisher Full Text\n\nCuéllar I, Roberts RE: Relations of depression, acculturation, and socioeconomic status in a Latino sample. Hisp J Behav Sci. 1997; 19(2): 230–8. Publisher Full Text\n\nKessler RC, Chiu WT, Demler O, et al.: Prevalence, severity, and comorbidity of 12-month DSM-IV disorders in the National Comorbidity Survey Replication. Arch Gen Psychiatry. 2005; 62(6): 617–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKerr DL, Santurri L, Peters P: A comparison of lesbian, bisexual, and heterosexual college undergraduate women on selected mental health issues. J Am Coll Health. 2013; 61(4): 185–94. PubMed Abstract \| Publisher Full Text\n\nGoldberg A, Kuvalanka K, Budge S, et al.: Health care experiences of transgender binary and nonbinary university students. Couns Psychol. 2019; 47(1): 59–97. Publisher Full Text\n\nMyers CA, Cowie H: Bullying at university: the social and legal contexts of cyberbullying among university students. J Cross Cult Psychol. 2017; 48(8): 1172–82. Publisher Full Text\n\nCook LJ: Striving to help college students with mental health issues. J Psychosoc Nurs Ment Health Serv. 2007; 45(4): 40–4. PubMed Abstract \| Publisher Full Text\n\nEisenberg D, Downs MF, Golberstein E, et al.: Stigma and help seeking for mental health among college students. Med Care Res Rev. 2009; 66(5): 522–41. PubMed Abstract \| Publisher Full Text\n\nStorrie K, Ahern K, Tuckett A: A systematic review: Students with mental health problems--a growing problem. Int J Nurs Pract. 2010; 16(1): 1–6. PubMed Abstract \| Publisher Full Text\n\nBlanco C, Okuda M, Wright C, et al.: Mental health of college students and their non-college-attending peers: results from the National Epidemiologic Study on Alcohol and Related Conditions. Arch Gen Psychiatry. 2008; 65(12): 1429–37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClement S, Schauman O, Graham T, et al.: What is the impact of mental health-related stigma on help-seeking? A systematic review of quantitative and qualitative studies. Psychol Med. 2015; 45(1): 11–27. PubMed Abstract \| Publisher Full Text\n\nStepakoff S: Effects of sexual victimization on suicidal ideation and behavior in U.S. college women. Suicide Life Threat Behav. 1998; 28(1): 107–26. PubMed Abstract\n\nTurchik JA: Sexual victimization among male college students: assault severity, sexual functioning, and health risk behaviors. Psychol Men Masc. 2012; 13(3): 243–55. Publisher Full Text\n\nSinozich S, Langton L: Rape and sexual victimization among college-aged females, 1995-2013. Bur Justice Statsitics US Dep Justice. 2014. Reference Source\n\nRAINN: Campus sexual violence: statistics. Rape Abus Incest Natl Netw. 2015. Reference Source\n\nCantor D, Fisher B, Chibnall S, et al.: Report on the AAU campus climate survey on sexual assault and sexual misconduct. Assoc Am Univ. 2015. Reference Source\n\nMartin B: Countering supervisor exploitation. J Sch Publ. 2013; 45(1): 74–86. Publisher Full Text\n\nNo place for bullies in science. Nature. 2018; 559(7713): 151. PubMed Abstract \| Publisher Full Text\n\nElse H: Does science have a bullying problem? Nature. 2018; 563(7733): 616–8. PubMed Abstract \| Publisher Full Text\n\nAnonymous: Academia is built on exploitation. We must break this vicious cycle. The Guardian. 2018. Reference Source\n\nYamada S, Cappadocia MC, Pepler D: Workplace bullying in Canadian graduate psychology programs: Student perspectives of student-supervisor relationships. Train Educ Prof Psychol. 2014; 8(1): 58–67. Publisher Full Text\n\nFrank E, Carrera JS, Stratton T, et al.: Experiences of belittlement and harassment and their correlates among medical students in the United States: Longitudinal survey. BMJ. 2006; 333(7570): 682. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKokkinos CM, Baltzidis E, Xynogala D: Prevalence and personality correlates of Facebook bullying among university undergraduates. Comput Hum Behav. 2016; 55(Part B): 840–50. Publisher Full Text\n\nSeguin E: Academic mobbing, or how to become campus tormentors. Univ Aff. 2016. Reference Source\n\nBrooks R: Student-parents and higher education: A cross-national comparison. J Ed Policy. 2012; 27(3): 423–39. Publisher Full Text\n\nPowell K: Why scientist-mums in the United States need better parental-support policies. Nature. 2019; 569(7754): 149–51. PubMed Abstract \| Publisher Full Text\n\nWoolston C: Feeling overwhelmed by academia? You are not alone. Nat Careers. 2018; 557: 129–131. Publisher Full Text\n\nPowell K: Work–life balance: Break or burn out. Nature. 2017; 545: 375–377. Publisher Full Text\n\nWoolston C: Psychology: Faking it. Nature. 2016; 529(7587): 555–7. PubMed Abstract \| Publisher Full Text\n\nParkman A: The imposter phenomenon in higher education: incidence and impact. J High Educ Theory Pract. (Ohio Dominican University U. 2016; 16(1): 51–60. Reference Source\n\nPowell K: Should we steer clear of the winner-takes-all approach? Nature. 2018; 553(7688): 367–9. PubMed Abstract \| Publisher Full Text\n\nServices UDSH and C: What is wellness? UC Davis. Reference Source\n\nGlobal Wellness Institute: History of wellness. 2019. Reference Source\n\nOswald AJ, Proto E, Sgroi D: Happiness and productivity. J Labor Econ. 2015; 33(4): 789–822. Publisher Full Text\n\nde Neve J, Diener E, Tay L, et al.: The objective benefits of subjective well-being. CEP Discuss Pap No 1236. 2013. Reference Source\n\nConley CS, Durlak JA, Dickson DA: An evaluative review of outcome research on universal mental health promotion and prevention programs for higher education students. J Am Coll Health. 2013; 61(5): 286–301. PubMed Abstract \| Publisher Full Text\n\nConley CS, Travers LV, Bryant FB: Promoting psychosocial adjustment and stress management in first-year college students: The benefits of engagement in a psychosocial wellness seminar. J Am Coll Health. 2013; 61(2): 75–86. PubMed Abstract \| Publisher Full Text\n\nUniversity of Illinois Counselling Centre: Suicide intervention policy. Univ Illinois Student Aff. 2015. Reference Source\n\nFernandez A, Howse E, Rubio-Valera M, et al.: Setting-based interventions to promote mental health at the university: a systematic review. Int J Public Health. 2016; 61(7): 797–807. PubMed Abstract \| Publisher Full Text\n\nZheng G, Lan X, Li M, et al.: Effectiveness of Tai Chi on Physical and Psychological Health of College Students: Results of a Randomized Controlled Trial. PLoS One. 2015; 10(7): e0132605. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCaldwell KL, Bergman SM, Collier SR, et al.: Effects of tai chi chuan on anxiety and sleep quality in young adults: lessons from a randomized controlled feasibility study. Nat Sci Sleep. 2016; 8: 305–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCaldwell K, Harrison M, Adams M, et al.: Developing mindfulness in college students through movement-based courses: Effects on self-regulatory self-efficacy, mood, stress, and sleep quality. J Am Coll Health. 2010; 58(5): 433–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVidic Z, Cherup N: Mindfulness in classroom: effect of a mindfulness-based relaxation class on college students’ stress, resilience, self- efficacy and perfectionism. Coll Stud J. 2019; 53(1): 130–144. Reference Source\n\nKeng SL, Smoski MJ, Robins CJ: Effects of mindfulness on psychological health: A review of empirical studies. Clin Psych Rev. 2011; 31(6): 1041–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBluth K, Campo RA, Pruteanu-Malinici S, et al.: A School-Based Mindfulness Pilot Study for Ethnically Diverse At-Risk Adolescents. Mindfulness (N Y). 2016; 7(1): 90–104. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuehner C, Huffziger S, Liebsch K: Rumination, distraction and mindful self-focus: Effects on mood, dysfunctional attitudes and cortisol stress response. Psychol Med. 2009; 39(2): 219–228. PubMed Abstract \| Publisher Full Text\n\nTimm C, Rachota-Ubl B, Beddig T, et al.: Mindfulness-Based Attention Training Improves Cognitive and Affective Processes in Daily Life in Remitted Patients with Recurrent Depression: A Randomized Controlled Trial. Psychother Psychosom. 2018; 87(3): 184–6. PubMed Abstract \| Publisher Full Text" }	[ { "id": "61928", "date": "14 Apr 2020", "name": "Teresa Evans", "expertise": [ "Reviewer Expertise Graduate trainee mental health", "K-12 STEM education", "neuroscience." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEach topic is discussed comprehensively with well-rounded information provided on the impacts of mental health on undergraduate, graduate, and postdoctoral fellows. Clarification is provided to ensure confusion is not made between discussion of undergraduate data and graduate level data, particularly via tables. Further, the authors provide clear definitions of Depression, Anxiety, and Wellness along with a discussion of the scales used to assess symptomatology. Discussion of challenges faced by graduate students and postdoctoral fellows is comprehensive and well referenced. It is also important to note that the authors provide a strong overview of the data present for medical and health students as well.\n\nAll statements provided are factual and supported by citations. The review is well written and accessible. Also, all conclusions drawn are appropriate for the literature cited and the state of the field.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] }, { "id": "62801", "date": "04 May 2020", "name": "Leland Ackerson", "expertise": [ "Reviewer Expertise Social Determinants of Health", "Social Epidemiology", "Intimate Partner Violence", "Global Health", "Mental Health." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe Introduction of the article points to the 2007 recession as a key event regarding the financial concerns of today’s students. I believe that updating this text to incorporate the Covid-19 related economic conditions will allow this article to remain relevant for a much longer time.\nSome of the cited papers actually diagnosed patients with mental illness (such as depression or anxiety). Others were epidemiologic studies which relied on psychometric tools that provided evidence of symptomology but did not actually diagnose patients. It may be helpful to make this distinction in the current review when relevant. For example, the Garlow et al. paper (reference #50) repeatedly refers to “depressive symptoms” while the relevant section of the current review (end of page 5 and beginning of page 6) refers to mild, moderate, and severe depression—not symptomology. I would encourage the authors to use language consistent with the referenced papers.\nBoth Liu et al. (reference #111) and Kerr et al. (reference #116) described sexual minorities as gay/lesbian. Yet on page 14 the authors used the term “homosexual” in the phrase “There is evidence that homosexual students experience poorer mental health and higher suicidality compared to heterosexual students111,116.” This term is outdated and should be replaced in that paragraph and the following paragraph.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-104
https://f1000research.com/articles/9-102/v1	10 Feb 20	{ "type": "Research Article", "title": "Effects of ‎tamoxifen on the reproductive system of female breast cancer patients: an ultrasound-based cohort study", "authors": [ "Ghasak Kais Abd-Alhussain‎", "Mohammed Qasim Yahya Mal-Allah Alatrakji‎", "Wieeam Abdulfattah Saleh‎", "Hayder Adnan Fawzi", "Aqeel‎ Shaker Mahmood‎", "Ghasak Kais Abd-Alhussain‎", "Mohammed Qasim Yahya Mal-Allah Alatrakji‎", "Wieeam Abdulfattah Saleh‎", "Aqeel‎ Shaker Mahmood‎" ], "abstract": "Background: Tamoxifen (TMX) is regarded as standard treatment for breast cancer (BC) patients‎. In recent years, several studies have reported gynecological side effects and due to TMX's estrogenic effects. Here, we evaluate the side effects of TMX on the ‎endometrium and ovaries of female BC patients. Methods: This was an ultrasound-based cohort study conducted in three oncology centers in Baghdad, Iraq. A total of ‎‎255 female patients were included, 140 premenopausal (PreM) and 115 postmenopausal (PostM), with estrogen receptor (ER)-positive BC using TMX adjuvant hormonal treatment for at least three months after surgery and adjuvant ‎chemo/radiotherapy.‎ Ultrasound (US) on the endometrium and ovaries of the women following ‎BC surgery/chemotherapy (baseline) and at 3, 6, 12, and 24 months following was performed‎. Data collected included age, menopausal status, co-morbid chronic illness and medications, including duration of TMX treatment. Results: Presence of ovarian cyst was significantly higher in the PreM ‎compared to PostM ‎women, while there were no significant differences for other gynecological findings.‎ At ‎baseline, endometrial thickness (ET) was significantly higher in the PreM compared to the PostM women. In both groups, women with increased ET became more frequent from baseline to 3 ‎months, from 3 to 6 ‎months, from 6 to 12 months, and from 12 ‎ to 24 months. At all time periods, ‎women with increased ET was ‎significantly higher in the PostM compared PreM women, resulting ‎in a risk of ET increase by 6 folds (ranging from 3 – ‎‎11 folds) ‎in PostM compared to PreM women. Conclusions: Longer duration of TMX is associated with increased ET. Duration of TMX did not appear to increase the risk of various gynecological outcomes, for example endometrial cancer rate was low. Finally, there was an increase in ET, which appeared to be six-folds higher in PostM compared to PreM women.‎", "keywords": [ "tamoxifen", "endometrial", "menopausal status", "gynecological side effect" ], "content": "Introduction\n\nBreast cancer (BC) is the second most prevalent cancer globally and the top cause of cancer-related deaths in women1. In Iraq, BC is the most prevalent cancer in the population. In 2012, BC occurred in 19.5% of all newly diagnosed cancer cases, and 34% of women with cancer2. Additionally, BC is the primary cause of cancer-related death in Iraqi women, causing 23.6% mortality among all women-related deaths).\n\nEstrogen receptors (ER) have multiple functions that affect the reproductive, musculoskeletal and cardiac systems, and the central nervous system. Additionally, it has been suggested to be involved in the proliferation of BC tumors tissues3; about 70–80% of BC cases are ER-positive. There are two classes of ER, α and β, and their activity is primarily regulated by estradiol (E2) binding. Therefore, E2 plays a vital role in the pathogenesis of BC through these receptors. Patients with these tumors are candidates for endocrine therapy after surgical removal of the primary tumor and treatment with ionizing radiation or cytotoxic chemotherapy4. The major strategy for BC treatment starts with surgery, chemotherapy, ionizing radiation therapy, endocrine (hormonal) therapy, and targeted therapy5.\n\nTamoxifen (TMX) is a selective ER modulator, which acts as an inhibitor for the effect of estrogen on breast tissues. Its side effects include vaginal dryness, vaginal discharge, flushes, cataracts, night sweats, thrombotic events, stroke, and rarely endometrial cancer and uterine cancer6. Many studies have examined the relationship between TMX use, duration of TMX use, whether female patients are premenopausal (PreM) or postmenopausal (PostM), and the development of gynecological side effects7–14. The present study aimed to identify if there is a relationship between TMX use and changes in the endometrium and ovaries of female breast cancer patients. In addition, the study aimed to classify if there are unreported gynecological side effects with TMX use, and whether menopausal status (PreM or PostM) leads to a different TMX effect on the endometrium and ovaries.\n\n\nMethods\n\nThis is an ultrasound (US)-based cohort study, conducted in three oncology centers In Bagdad, Iraq. Female BC patients treated with TMX at dose of 20 mg/day as part of therapeutic regimen for the treatment of BC were included in the study. Abdominal US was used for the assessment of endometrial thickness (ET) and abnormalities in the endometrial cavity and ovaries.\n\nAll procedures performed in the study were in accordance with the ethical standards of the Institutional Research Committee at the College of Medicine, Baghdad University, who approved the study protocol (approval date: 3rd December 2019; number: 2019/0234), and with the 1964 Helsinki declaration and its later amendments.\n\nWritten informed consent was obtained from all participants to be included in the study.\n\nThe study was conducted at the three main oncology centers in Baghdad: Breast Cancer Center of Al-Elwia Teaching Hospital, Oncology Teaching Hospital at Baghdad Medical City, and Al-Amel Oncology Hospital. These oncology centers are teaching hospitals under the supervision of the College of Medicine, Baghdad University. Periods of recruitment were between December 2018 and May 2019. Data about patients (US findings) were obtained from their hospital records (each patient was followed-up for at least 6 months, with maximum duration of follow-up of 2 years).\n\nA sample size of 256 women was calculated considering the power of 80%, the confidence level of 95% and a relative precision 5% and prevalence of ET of 12% in PreM and 10.6% in PostM based on a study by Lee et al. 201815. Therefore, a 20% value was chosen, the following formula were used to calculate the estimated sample size:\n\n\n\nWhere, p is prevalence, q is (1 – p), and d is relative precision.\n\nA total of 255 patients, 140 premenopausal and 115 postmenopausal female BC patients were included in the study.\n\nWe included female patients with ER-positive BC treated with TMX as an adjuvant hormonal treatment at a dose of 20 mg/day for at least three months after surgery and in addition to adjuvant chemo/radiotherapy.\n\nWe excluded patients who were on irregular treatment, patients with increased uterine thickness for any reason at baseline assessment, patients who had other gynecological or non-gynecological malignancies, patients with secondary BC and patients with incomplete or lost medical records.\n\nHormonal levels and menstrual history were used for the assessment of menopausal status. A PreM woman was defined as a woman with regular menstrual cycles with follicular stimulating hormone (FSH) <40.0 mIU/mL. A PostM woman defined as a woman with amenorrhea longer than one year and FSH >40 mIU/mL on two sequential occasions at the time of diagnosis with BC15.\n\nAll women that participated in the study were interviewed by the researchers; data were collected from them using a predesigned survey in a personal interview setting and from their medical records. The women were followed-up prospectively, and if the women had previous US reading it will be incorporated in study. Patient characteristics, US findings, and results of histopathological examination of endometrial biopsy whenever available were collected. The survey collected the following patient characteristics: age of the patient, menopausal status, co-morbid chronic illness and past medical history (e.g. hypertension, diabetes mellitus (DM)), duration of TMX use, side effects, and current used medications.\n\nAbdominal US was used for the assessment of ET and abnormalities in the endometrial cavity and ovaries. Endometrial and ovarian assessment by US was performed before the start of hormonal therapy and alterations were periodically measured at an interval of three months. Assessment of the endometrial lining was done by calculating the maximum thickness from the outermost limits of the endometrial-myometrial juncture and the thickening was defined according to menopausal status as follows16:\n\n- PreM women with endometrial thickness ≥12 mm;\n\n- PostM women with endometrial thickness ≥ 5 mm.\n\nPatients who were diagnosed with endometrial pathology were followed-up for six months to assess their status and dealt with accordingly. Patients diagnosed with ovarian cyst (> 30 mm on US) were further evaluated by cancer antigen 125 (CA-125); if normal, US examination was commenced every three months, if high CA-125, patients underwent further radiological assessment, including CT scan or MRI15.\n\nDiagnostic hysteroscopy was done for the patients that showed abnormalities on US and biopsy was taken whenever indicated (when the US demonstrated uterine abnormalities or mass, and/or patients with abnormal uterine bleeding).\n\nChi-square test, Fisher’s exact test, independent t-test, and paired t-test were used to compare between PreM and PostM groups. Logistic regression used to calculate the odds ratio (OR) and 95% confidence intervals. Linear regression analysis was performed to assess the relationship between different variables. SPSS 23.0.0 (Chicago, IL) and GraphPad Prism version 8.0.0 used to perform statistical analysis. P<0.05 was considered significant.\n\n\nResults\n\nIn total, 255 women with BC were included in this study. Age range was 32 to 78 years, with mean age 50.4±9.0 years. The most common age group was 40–49 years (36.1%), followed by 50–59 years (32.5%). In total, 15.3% of the patients had DM, while 3.9% had hypertension (a higher frequency in the PreM compared to PostM women).\n\nThere was no significant difference in the history of hypertension, DM, use of medications (angiotensin-converting enzyme/angiotensin receptor blocker, or metformin), cancer stage, and treatment duration of TMX between PreM and PostM women (Table 1).\n\nACE: angiotensin-converting enzyme, ARB: angiotensin receptor blocker, TMX: tamoxifen\n\nFrom US examinations, only the presence of ovarian cyst was significantly higher in the PreM compared to PostM women. Other US findings were not significant between groups (Table 2).\n\nAt baseline, ET was significantly higher in the PreM compared to the PostM group. In both groups, women with increased ET became more frequent from baseline to 3 months, from 3 months to 6 months, from 6 months to 12 months, and from 12 months to 24 months. At all time periods, the number of women with increased ET was significantly higher in the PostM compared with PreM group (Table 3; Figure 1).\n\n# PreM women with ET ≥12 mm, PostM women with ET ≥5 mm considered a positive finding.\n\nET was significantly higher in PostM compared to PreM women, resulting in an increased risk of endometrial thickening by 6 fold (OR: 6.000, 95%CI: 3.313 – 10.867, p-value <0.001) in PostM compared to PreM women (Figure 2).\n\nAt baseline there was not a significant correlation between duration of TMX with ET; however from 3 months until 24 months after TMX therapy there was significant correlation between duration of TMX with EM thickness (Table 4).\n\nr: correlation coefficient\n\nThere was no significant correlation between TMX treatment duration with any gynecological outcomes (Table 5).\n\nOR: odds ratio, CI: confidence interval\n\n\nDiscussion\n\nIn the present study, mean ET after 24 months was 11.1±6.0 mm in PreM and 13.0±9.0 mm for PostM women (no significant difference between groups). This agreed with other studies17,18. In a retrospective study that examined 614 women with BC, 53 of them had history of TMX usage, and ET was significantly higher in women that received TMX (11 vs 6 mm in those not on TMX therapy). In addition, women with ET ≥5 mm was significantly higher in TMX group (86.8% vs. 52.0%, p-value <0.001), and higher than other endometrial abnormalities (43.4% vs 31.7%, p-value = 0.048), which indicates that the use of TMX increases the risk of ET and abnormalities18.\n\nIn the current study, increased ET occurred in 45.7% of PreM and 83.5% of PostM women, and these findings were similar to other studies17. However, other studies reported a lower rate of increased ET than the present study; Buijs et al. examined 47 PreM women and found that 7 (17.0%) suffered from increased ET (≥12 mm)19. Jindal et al. reported that 30% of women assessed had EM thickness ≥5 mm which is lower than our study20. Another study of 737 PostM patients with BC who received TMX, revealed that 28% had ET ≥6 mm21, while Lee et al. reported an increased ET in 12% of PreM and 10.6% of PostM women15.\n\nIn the current study, there was a significant relationship between TMX treatment duration and ET (the magnitude of this relationship was similar from 3 months to 24 months). In a study by Hann et al., ET increased with the duration of TMX treatment; 73 women treated with TMX for <5 years had a median ET of 5 mm, and 44% of biopsies yielded abnormal results, while 18 women who had received TMX for ≥5 years had a median ET of 14 mm (58% of endometrial biopsies in this group were abnormal)22. This study agrees with our findings. In contrast, Jindal et al. reported no significant correlation between TMX duration and ET, which could be attributed limitations of their study, including a small sample size and short follow-up duration21.\n\nEndometrial polyps are a common pathology and have an increased malignant transformation rate in PostM TMX-treated BC patients17; however, limited studies have investigated the malignant potential of endometrial polyps by hysteroscopy in this population17. In the present study, endometrial polyps were present in 2.1% of PreM women and 6.1% of PostM women. In Jeon et al., the frequency of endometrial polyps was much higher than our findings (41.7%)17. Similarly, Hann et al. found endometrial polyps in 33% cases22 and Deligdisch et al. found a frequency of 23.14%23. Abdaal et al. revealed similar rate of polyps to the present study of 3.9%18. The low incidence of EM polyps in the present study compared with other studies could suggest that the incidence in Iraqi women is lower than other ethnicities. In the present study, endometrial hyperplasia in PreM and PostM women was 5.7% and 11.3%, respectively, while it was 1.7% in Jeon et al.17 and 8% in Deligdisch et al.23.\n\nThe use of TMX is associated with two to four fold increased risk of endometrial hyperplasia and polyps24. The increased risk of these complications is related to the effect of TMX on the endometrium, which causes proliferation of the endometrium, particularly in PreM and early PostM women25. In the present study, endometrial cancer occured in 4.3% and 5.3% of PreM and PostM women, respectively, and these findings are similar to other studies18,22,23, apart from Jeon et al.17. In the present study, there was no significant relationship between duration of TMX and risk of endometrial cancer (OR: 0.994, 95%CI: 0.675-1.463), which was in agreement with Katase et al., who concluded that TMX did not increase the risk of endometrial cancer in women with primary BC8. However, another study showed that endometrial cancer was related to TMX and the risk of endometrial cancer is related to the duration of TMX use26. This also confirmed by a meta-analysis7.\n\nAnother significant finding in the present study was that the presence of ovarian cyst was significantly higher in PreM (27.9%) compared to PostM women (7.8%), which is in agreement with other studies15,24.\n\nThe retrospective nature of the study is a potential bias. In addition, the short duration of prospective follow-up (i.e. six months) also added limitation to the number of events observed the researcher.\n\n\nConclusions\n\nLonger duration of TMX is associated with increased ET in Iraqi women with BC; however, the duration of TMX did not appear to increase the risk of various gynecological outcomes. In addition, endometrial cancer rate was low, and there was a high rate of ET, which appears to be six-folds higher in PostM compared to PreM women.\n\n\nData availability\n\nZenodo: Effects of Tamoxifen on the Reproductive System of Females with Breast Cancer, http://doi.org/10.5281/zenodo.357622227.\n\nThis project contains the following underlying data:\n\n- Extended Data File data setting.xlsx (raw data for all 255 women interviewed)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nSiegel R, Naishadham D, Jemal A: Cancer statistics, 2012. CA Cancer J Clin. 2012; 62(1): 10–29. PubMed Abstract \| Publisher Full Text\n\nAlwan NAS: Breast Cancer Among Iraqi Women: Preliminary Findings From a Regional Comparative Breast Cancer Research Project. J Glob Oncol. 2016; 2(5): 255–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDutertre M, Smith CL: Molecular mechanisms of selective estrogen receptor modulator (SERM) action. J Pharmacol Exp Ther. 2000; 295(2): 431–7. PubMed Abstract\n\nAli S, Coombes RC: Estrogen receptor alpha in human breast cancer: occurrence and significance. J Mammary Gland Biol Neoplasia. 2000; 5(3): 271–81. PubMed Abstract \| Publisher Full Text\n\nMatsen CB, Neumayer LA: Breast cancer: a review for the general surgeon. JAMA Surg. 2013; 148(10): 971–9. PubMed Abstract \| Publisher Full Text\n\nOlver IN: Prevention of breast cancer. Med J Aust. 2016; 205(10): 475–9. PubMed Abstract \| Publisher Full Text\n\nMacMahon B: Overview of studies on endometrial cancer and other types of cancer in humans: perspectives of an epidemiologist. Semin Oncol. 1997; 24(1 Suppl 1): S1-122–S1-39. PubMed Abstract\n\nKatase K, Sugiyama Y, Hasumi K, et al.: The incidence of subsequent endometrial carcinoma with tamoxifen use in patients with primary breast carcinoma. Cancer. 1998; 82(9): 1698–703. PubMed Abstract \| Publisher Full Text\n\nJones ME, van Leeuwen FE, Hoogendoorn WE, et al.: Endometrial cancer survival after breast cancer in relation to tamoxifen treatment: pooled results from three countries. Breast Cancer Res. 2012; 14(3): R91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJolles CJ, Smotkin D, Ford KL, et al.: Cystic ovarian necrosis complicating tamoxifen therapy for breast cancer in a premenopausal woman. A case report. J Reprod Med. 1990; 35(3): 299–300. PubMed Abstract\n\nSherman BM, Chapler FK, Crickard K, et al.: Endocrine consequences of continuous antiestrogen therapy with tamoxifen in premenopausal women. J Clin Invest. 1979; 64(2): 398–404. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchwartz LB, Krey L, Demopoulos R, et al.: Alterations in steroid hormone receptors in the tamoxifen-treated endometrium. Am J Obstet Gynecol. 1997; 176(1 Pt 1): 129–37. PubMed Abstract \| Publisher Full Text\n\nAnzai Y, Holinka CF, Kuramoto H, et al.: Stimulatory effects of 4-hydroxytamoxifen on proliferation of human endometrial adenocarcinoma cells (Ishikawa line). Cancer Res. 1989; 49(9): 2362–5. PubMed Abstract\n\nLove CD, Muir BB, Scrimgeour JB, et al.: Investigation of endometrial abnormalities in asymptomatic women treated with tamoxifen and an evaluation of the role of endometrial screening. J Clin Oncol. 1999; 17(7): 2050–4. PubMed Abstract \| Publisher Full Text\n\nLee S, Kim YH, Kim SC, et al.: The effect of tamoxifen therapy on the endometrium and ovarian cyst formation in patients with breast cancer. Obstet Gynecol Sci. 2018; 61(5): 615–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMavaddat N, Michailidou K, Dennis J, et al.: Polygenic Risk Scores for Prediction of Breast Cancer and Breast Cancer Subtypes. Am J Hum Genet. 2019; 104(1): 21–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJeon SJ, Lee JI, Lee M, et al.: Endometrial polyp surveillance in premenopausal breast cancer patients using tamoxifen. Obstet Gynecol Sci. 2017; 60(1): 26–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdaal A, Mushtaq Y, Khasati L, et al.: Post-menopausal bleeding - Is transvaginal ultrasound a useful first-line investigation in tamoxifen users? Post Reprod Health. 2018; 24(2): 72–8. PubMed Abstract \| Publisher Full Text\n\nBuijs C, Willemse PH, de Vries EG, et al.: Effect of tamoxifen on the endometrium and the menstrual cycle of premenopausal breast cancer patients. Int J Gynecol Cancer. 2009; 19(4): 677–81. PubMed Abstract \| Publisher Full Text\n\nJindal A, Mohi MK, Kaur M, et al.: Endometrial evaluation by ultrasonography, hysteroscopy and histopathology in cases of breast carcinoma on Tamoxifen therapy. J Midlife Health. 2015; 6(2): 59–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCohen I, Rosen DJ, Tepper R, et al.: Ultrasonographic evaluation of the endometrium and correlation with endometrial sampling in postmenopausal patients treated with tamoxifen. J Ultrasound Med. 1993; 12(5): 275–80. PubMed Abstract \| Publisher Full Text\n\nHann LE, Giess CS, Bach AM, et al.: Endometrial thickness in tamoxifen-treated patients: correlation with clinical and pathologic findings. AJR Am J Roentgenol. 1997; 168(3): 657–61. PubMed Abstract \| Publisher Full Text\n\nDeligdisch L, Kalir T, Cohen CJ, et al.: Endometrial histopathology in 700 patients treated with tamoxifen for breast cancer. Gynecol Oncol. 2000; 78(2): 181–6. PubMed Abstract \| Publisher Full Text\n\nKim HS, Jeon YT, Kim YB: The effect of adjuvant hormonal therapy on the endometrium and ovary of breast cancer patients. J Gynecol Oncol. 2008; 19(4): 256–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDecensi A, Fontana V, Bruno S, et al.: Effect of tamoxifen on endometrial proliferation. J Clin Oncol. 1996; 14(2): 434–40. PubMed Abstract \| Publisher Full Text\n\nBernstein L, Deapen D, Cerhan JR, et al.: Tamoxifen therapy for breast cancer and endometrial cancer risk. J Natl Cancer Inst. 1999; 91(19): 1654–62. PubMed Abstract \| Publisher Full Text\n\nFawzi HA: Effects of Tamoxifen on the Reproductive System of Females with Breast Cancer – an Ultrasound-based Cohort study (Version Microsoft Excel Worksheet) [Data set]. Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3576222" }	[ { "id": "64076", "date": "09 Jun 2020", "name": "Marian J E Mourits", "expertise": [ "Reviewer Expertise My field of expertise is gynecological oncology", "with special focus on hereditary gynecological cancer. I have a PhD in the field of gynecological side effects of tamoxifen." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study is on gynecological side effects of adjuvant tamoxifen in Iraqi women after breast cancer treatment.\nAim of the study is to classify unreported gynecological side effects and whether menopausal status leads to differential effects.\nThis aim has been studied before, however the authors do not seem to be familiar with earlier studies on the differential gynecological side effects of tamoxifen.\nSome more detailed comment are given:\nINTRODUCTION\nThe pharmacological background of the side effects in the introduction is not sufficient. The authors need to describe the differential side effects of tamoxifen depending of the estrogen level of the patient.\n\nIn premenopausal women tamoxifen acts as an anti-estrogen, an estrogen antagonist, on the endometrium [comparable as clomifen citrate] and as ovulation induction on the ovaries, causing multiple ovarian cysts. This often causes amenorrhoea in premenopausal women, which can be misinterpreted as postmenopause, if last menstrual cycle is taken as a measure. However, these ovarian cysts produce high levels of estradiol [therefor these women have no hot flushes although they have no menstrual periods].\n\nReferences Mourits MJ, de Vries EG, ten Hoor KA, van der Zee AG, Willemse PH.Mourits MJ, et al. J Clin Oncol. 2007 Aug 20;25(24):3787-8; author reply 3788-9. doi: 10.1200/JCO.2007.11.1633.J Clin Oncol. 2007. PMID: 17704431 1 Beware of amenorrhea during tamoxifen: it may be a wolf in sheep's clothing. Ovarian tumors in postmenopausal breast cancer patients treated with tamoxifen I Cohen, Y Beyth, R Tepper, et al. Gynecol Oncol, 60 (1996), pp. 54-58 2 MJE Mourits, EGE De Vries, PHB Willemse, et al.Ovarian cysts in women receiving tamoxifen for breast cancer Br J Cancer, 79 (1999), pp. 1761-1764 3\n\nIn the low-E2 environment in postmenopausal women, tamoxifen acts as an estrogen- agonist on the endometrium. This results in thickened endometrium after long term use. However, this ET is not caused by proliferated endometrial epithelium, but by thickened stroma. Therefore, endometrial biopsies in postmenopausal women with enlarged ET usually result in little if any endometrial tissue. Not the epithelium, but the stroma is enlarged.\n\nReference Discrepancy between ultrasonography and hysteroscopy and histology of endometrium in postmenopausal breast cancer patients using tamoxifen. Mourits MJ, Van der Zee AG, Willemse PH, Ten Hoor KA, Hollema H, De Vries EG.Mourits MJ, et al. Gynecol Oncol. 1999 Apr;73(1):21-6. doi: 10.1006/gyno.1998.5316.Gynecol Oncol. 1999. PMID: 10094875 4\nSTUDY DESIGN\nIt is not clear what the primary outcome measure is.\n\nThe authors aim to classify 'unreported gynecological side effects' and ' whether menopausal status leads to differential effects'.\n\nQuestions:\nWhat would than be their outcome measure and with what consequences? ET?, for what reason would ET be interesting, because it is not a diseases, and does not need to be treated. Gynecological abnormalities? Ovarian cysts?\n\nIf the aim is to classify 'unreported' gynecological side effects, the authors should give an extensive review about 'what has been reported' to know what is 'unreported'.\n\nThe rationale of the sample size calculation is not clear to me.\n\nWhat is meant by a 'prevalence of ET' [endometrial thickness] of 12% in pre- and 10.6% in postmenopausal women, to prove what?\n\nWhat was defined as endometrial pathology?\n\nIt is not clear whether the study is a prospective cohort study - which is suggested by the study design description and recruitment of new[?] patients between Dec 2018 until May 2019 - or a retrospective study - which is written in the Study Limitations paragraph - 'The retrospective nature of the study is a potential bias.' Thi is very confusing.\n\nAnother problem is that it is not clearly described at what moment the baseline US was performed. Was T0/baseline before starting tamoxifen? How long after chemotherapy?\n\nThere is a problem with the definition 'premenopausal' and 'postmenopausal'. As many young women on tamoxifen have no menstrual periods while on medication due to anovulation through hyperstimulated ovarian cysts. This issue has not been addressed properly.\n\nHow many women were on LHRH antagonists?\n\nWomen needed to be on tamoxifen for at least three months, a follow up at 3, 6, 12 and 24 months was performed / reported. However it is not clear how many women were followed up for the total duration of the study.\n\nNeither is the end of the study in time reported.\nRESULTS\nThe authors report on endometrial thickness [ET] but do not report on the 'Swiss cheese' aspect of the postmenopausal endometrium after long term tamoxifen exposure.\n\nQuestion - did they not observe this, or did they not report on it?\n\nThe authors do report on ' the risk of ET', however ET is not a disease.\n\nQuestion - what do the authors mean by ' risk of ET'?\nDISCUSSION\nIn the discussion, the authors do not report separately the literature on pre- and postmenopausal women, which needs to be done, given the differential effects of tamoxifen in these both groups.\n\nThe authors miss some important studies many years ago, which addressed these issues on a more causal and explanatory level. These need to be studied for a comprehensive discussion.\nIN CONCLUSION\nAlthough the authors did a great job in performing many US, in many breast cancer patients, this study does not add to the knowledge and understanding of tamoxifen effects on the female genital tract, due to the above mentioned comments. Only thorough re-writing of this paper with a clear clinical aim, could make it worthwhile publishing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "64074", "date": "22 Jun 2020", "name": "Lisa Rydén", "expertise": [ "Reviewer Expertise Surgical oncology (hormonal therapy of endocrine-responsive breast cancer)" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments\nThis is an observational study not reported according to the STROBE criteria and lacks a flow-chart explaining inclusion and exclusion criteria.\n\nNo pre-specified end-point is provided other than “changes in the endometrium and ovaries of female breast cancer patients”. An end-point has to be clearly defined and measurable.\n\nThe sample size is difficult to follow including the prevalence of endometrial thickness but not any pre-specified hypothesis on change of thickness in relation to TAM therapy. The sample size calculations seems to be based on the difference between pre- and postmenopausal patients, not any effect by TAM.\n\nAbdominal ultrasound for measuring endometrial thickness is not state-of the-art which makes it difficult to compare the results to previous publications. If abdominal ultrasound is the method of choice in Iraq it would have been interesting to add data on its performance in relation to transvaginal assessments.\n\nThe availability of abdominal ultrasound for BC patients is not presented nor is there any data on any additional interventions caused by this ambitious ultrasound program. Are all BC patients on TAM offered abdominal ultrasound in Iraq? And if not, is there enough data from this study to recommend/abstain it as a routine surveillance.\nSpecific questions to be addressed when assessing original research papers\nIs the work clearly and accurately presented and does it cite the current literature?\nNo, it does not adhere to the guidelines of presenting observational studies (see p1-3) and the reference list is outdated (far too old publications).\n\nIs the study design appropriate and is the work technically sound?\nSee p 1-3\n\nAre sufficient details of methods and analysis provided to allow replication by others?\nI have not found any report on abdominal US so this makes it difficult to replicate the study, albeit the method might be appropriate if data on its accuracy compared to transvaginal US could be provided – either as a reference or by own data.\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nSee sample size comments, otherwise I can´t assess it.\n\nAre all the source data underlying the results available to ensure full reproducibility?\nOK\n\nAre the conclusions drawn adequately supported by the results?\nThe conclusion is sound but should clearly state that intense surveillance of ET by US doesn´t seem to prevent any gynaecological disorders and thus intensive follow-up schemes has no place as a clinical routine.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-102
https://f1000research.com/articles/9-98/v1	10 Feb 20	{ "type": "Study Protocol", "title": "Impact evaluation of the Steno REACH Certificate Course in Clinical Diabetes Care for health care providers in Malaysia: protocol for a quasi-experimental, mixed-methods research study", "authors": [ "Feisul Mustapha", "Michael Calopietro", "Karoline Kragelund Nielsen", "Jens Aagaard-Hansen", "Shiang Cheng Lim", "Ulla Bjerre-Christensen", "Michael Calopietro", "Karoline Kragelund Nielsen", "Jens Aagaard-Hansen", "Shiang Cheng Lim", "Ulla Bjerre-Christensen" ], "abstract": "The burden of diabetes continues to increase in Malaysia, and the public primary health sector has an insufficient number of health care providers well-trained in diabetes care. The Ministry of Health Malaysia collaborated with Steno Diabetes Center to educate primary care doctors and nurses on the fundamentals of clinical diabetes care using a competency-based approach that blends e-learning, classroom-based learning, and clinic-based group work. This programme is called Steno REACH Certificate Course in Clinical Diabetes Care (SRCC).\n\nThe aim of this study was to assess the effectiveness of the SRCC intervention in improving diabetes-related knowledge, attitudes, skills and clinical practices among non-specialised doctors and general nurses working in public health clinics in Malaysia. This paper presents the study protocol.\n\nA quasi-experimental, mixed-methods study based on Solomon’s Four Group Design was applied. Non-specialist doctors and general nurses from ten health clinics were randomly selected to receive the educational intervention. Comparison clinics were purposive selected matching on proxy indicators for quality of diabetes care. The intervention consisted of 50 hours of e-learning, 48 hours of classroom-based learning and approximately 25 hours of work-based learning that covered all main aspects of clinical diabetes care and delivered over a six-month period. Primary outcomes were changes in diabetes-related knowledge, attitudes, skills, and clinical practice. Patients’ perceptions regarding the quality of care provided were classified as a secondary outcome. Other outcome measures included patients' assessment of their chronic disease care and providers' perceptions, attitudes and perceived barriers in care delivery.\n\nResults from this study will inform future educational approaches within the Malaysian health system. The study is unique because it evaluated a pertinent public health topic using a very robust methodology.", "keywords": [ "Continuing medical education", "diabetes", "healthcare providers", "Malaysia", "mixed methods", "Solomon’s Four Group Design" ], "content": "Introduction\n\nThe burden of diabetes continues to increase in Malaysia. Data from the 2015 population-based National Health and Morbidity Survey (NHMS) showed a prevalence of 17.5% among adults aged 18 years and above, which translated to about 3.5 million adults1. The overall prevalence of diabetes is amongst the highest in the Asia-Pacific region, excluding the pacific island nations2.\n\nPrimary health care is at the center of the Malaysian healthcare system and is supported by secondary and tertiary care. Malaysia has a well-established, public primary health care structure, with 1,061 health clinics, 1,810 community clinics, and 307 “out-reach” health clinics (called 1Malaysia clinics)3. Although Malaysia has a parallel public and private system, the majority of treatment for chronic diseases is provided by the public health system which is heavily subsidised by the government.\n\nThe Malaysian healthcare delivery system is facing increasing pressure to provide quality care to patients with diabetes. The latest data showed that about 80% of diagnosed diabetes patients seek treatment at MOH healthcare facilities and this proportion is expected to continue to increase1. As the burden of diabetes increases, the public health sector is faced with an insufficient number of well-trained diabetes medical practitioners to handle the increasing number of diabetes patients4. This has contributed to a treatment gap that requires MOH healthcare professionals to be better equipped to provide patient-centered diabetes services at the primary care level.\n\nA large part of addressing this treatment gap focuses on human resource capacity building in the management of non-communicable diseases (NCDs)5,6. Continuing medical education (CME) is a cornerstone in developing clinical skills and ensuring high-quality patient care by nurses and medical doctors. Cervero and Gaines (2015) conclude in a synthesis of systematic reviews, that CME improves physician performance and patient outcomes7, and while studies on CME in general have reported a positive effect on physician performance and patient outcomes, several have expressed the need for further studies on the implementation of knowledge, skills, and attitudes, as well as the impact of contextual and implementation factors7. Despite the importance of CME, few studies have measured the clinical impact of CME on diabetes in a real-world setting8–10. These studies have shown varying results8,10–13. Studies have also shown that CME is associated with increased satisfaction and a better psychosocial wellbeing of diabetes patients11, and that it is very well received among participating healthcare providers (HCPs)14.\n\n\nThe SRCC intervention\n\nAs part of its efforts to build diabetes management capacity, MOH Malaysia collaborated with Steno Diabetes Center to educate primary care doctors and nurses on the fundamentals of clinical diabetes care using a competency-based approach that blends e-learning, classroom-based learning, and clinic-based group work. This programme is called the Steno REACH Certificate Course in Clinical Diabetes Care (SRCC). The goal of SRCC is to improve the knowledge and skills of participating HCPs in clinical diabetes management, thereby empowering them to provide high-quality diabetes care. The course was designed for primary care doctors and nurses with little training in diabetes care and was facilitated by a team of Malaysian health care professionals trained to deliver the programme by experts from Steno Diabetes Center Copenhagen.\n\nThe curriculum included ten modules:\n\n1. Diagnosis and Pathophysiology\n\n2. Patient Engagement\n\n3. Non-Pharmacological Treatment\n\n4. Pharmacological Treatment\n\n5. Insulin Therapy\n\n6. Acute Complications\n\n7. Microvascular Complications\n\n8. Macrovascular Complications\n\n9. Diabetes and Pregnancy\n\n10. Clinical Quality\n\nTwo versions of each module were developed, one for doctors and one for nurses, with content tailored to their individual job roles. The intervention was designed to have learners cycle through a schedule of approximately 50 hours of independent online study and 48 hours of face-to-face classroom time. The e-learning content included all foundational materials delivered in an interactive learning environment. Participants completed the first five modules before coming to the classroom to reinforce key learning outcomes through interactive learning activities. This experience repeated itself for modules 6 to 10. In addition, participants were assigned work-based learning activities to complete in the periods between classroom-based sessions. Work-based learning activities included defining and reflecting on personal learning goals, patient journaling, article discussions with clinic peers, case discussions with clinic peers, and medical record reviews with clinic leaders. Together, work-based learning activities took an additional 25 to 30 hours of learning time.\n\nThe intervention was piloted twice between 10 October 2015 and 4 December 2016 to ensure its proper functioning and make any necessary curriculum improvements. Pilot course participants were recruited using a clinic-based recruitment approach. Ten interested clinics were invited to send at least one doctor and one nurse in order to reinforce the team-based care model essential to good diabetes clinical care. A total of 22 non-specialist doctors and 40 general nurses participated in the pilot studies.\n\nThis paper describes the protocol of the impact evaluation of the SRCC.\n\n\nStudy objectives\n\nThe impact evaluation aims to assess the effect of participation of non-specialised medical doctors and general nurses in the SRCC in selected public health clinics in Malaysia.\n\n(1) To measure changes in diabetes-related knowledge, attitudes and clinical skills before and after course participation;\n\n(2) To assess whether course participation and improved diabetes-related knowledge and clinical skills translate into changes in an individual’s clinical practice; and\n\n(3) To examine the influence of contextual factors at facility, health system and sociocultural levels on individual diabetes-related clinical practice changes.\n\nThe primary hypothesis of this research was that participation in the SRCC will result in increased knowledge about diagnosing diabetes, the role of diet and exercise in the treatment of diabetes, oral and injectable therapies used in the treatment of diabetes, common diabetes complications, and approaches to patient engagement. It was also the hypothesis that improved knowledge would translate into improved clinical skills in these aspects of care and that providers’ attitudes about people with diabetes would be positively impacted.\n\nIn addition, it was hoped that 1) patients’ perceptions of their care experience would improve with the intervention, and 2) primary care providers’ perceptions, attitudes, experiences and perceived barriers in implementing the intervention were explored.\n\n\nMethods\n\nA quasi-experimental, mixed-methods approach was applied in this study, based on Solomon’s Four Group Design, which is ideally used in evaluation of educational interventions that contain pre- and post-assessments. This design is recommended when it is possible that the pre-test could influence later tests, for example by learning or priming effects. This design contain four groups. In addition to the basic pre-test/intervention/post-test groups, three additional groups are included: one that received both tests, but not the intervention; one that received the intervention without the pre-test only; and one with neither pre-test nor intervention. This design serves to reduce the influence of confounding variables and allows the researcher to test whether the pretest itself has an effect on the subjects. Doctors and nurses from comparison clinics not participating in SRCC were also assessed to isolate individual and clinic level changes that may be attributable to the educational intervention.\n\nEach of the four study arms were subjected to a different set of data collection methods at different points of the intervention. This is shown diagrammatically in Figure 1.\n\nFrom a pre-determined sampling frame, health clinics in the states of Kuala Lumpur and Selangor were randomly selected for the four arms of this research. These two states were selected as they have the full range of the diverse types of health clinics located in Malaysia. The four arms were selected to conform to Solomon’s Four Group Design. This design allows researchers to control for the possibility of a test-effect.\n\nArm 1: Intervention clinics with pre and post investigations\n\nArm 2: Intervention clinics with post investigation only\n\nArm 3: Comparison clinics with pre and post investigations (no intervention)\n\nArm 4: Comparison clinics with a single investigation only (no intervention)\n\nThe pilot test of the educational intervention included ten clinics, and based on this experience, ten clinics were deemed an appropriate number of clinics to support a diverse learning experience among participants while still limiting class size to an appropriate level for delivering a heavily facilitated and interactive learning experience. All MOH health clinics in Selangor and Kuala Lumpur meeting the inclusion criteria were subjected to a two-stage stratified random sampling for selection of the ten intervention clinics. Clinics meeting the following criteria were eligible for random selection:\n\nInclusion criteria were: MOH health clinics with more than 1,000 registered active diabetes patients (as defined and registered in the National Diabetes Registry).\n\nExclusion criteria were: MOH health clinics that have participants enrolled in the two SRCC pilot classes.\n\nA total of 70 possible clinics were reduced to 43 eligible clinics based on the above criteria, and these 43 were included in the stratified, random selection process. For the first stage, the 43 health clinics were divided into the following three categories based on the number of registered active diabetes patients:\n\nBetween 1,000 and 1,999 patients\n\nBetween 2,000 and 2,999 patients\n\n≥3,000 patients\n\nFor the second stage the health clinics were divided into the following two categories based on the variety and complexity of medical services they provide as defined by MOH:\n\nIntermediate clinic\n\nAdvanced clinic\n\nBased on the above criteria, the number of eligible clinics is summarised in Table 1. Ten clinics were then randomly selected for inclusion as the intervention clinics as shown in Table 2.\n\nThese ten clinics were then randomly allocated to one of the two intervention arms. Six clinics were allocated to Arm 1 and four were allocated to Arm 2. The six-to-four split was to allow for more pre- and post-data to be included for the intervention arm of research, as per Solomon’s Four Group Design, only Arm 1 of the intervention clinics has both pre- and post-data available.\n\nComparison clinics were then selected from the remaining post-stratification clinics. Six comparison clinics were selected for Arm 3 using a purposive approach in which comparison clinics were matched based on a proxy indicator for quality of diabetes care. Using data from the National Diabetes Registry (NDR), the mean HbA1c value for all patients with diabetes in each intervention clinic was calculated and a comparison clinic with the closest mean HbA1c value for all diabetes patients was matched and selected for inclusion in Arm 3. The same process was used to select the four comparison clinics in Arm 4, which were chosen to match the four intervention clinics in Arm 2.\n\nFrom each of the ten health clinics selected to receive the intervention, the participants were chosen by the Family Medicine Specialists (FMS) who are the clinical heads of the health clinic, based on the following criteria:\n\n(1) Each clinic nominated at least four participants, two medical officers and two nurses.\n\n(2) The medical officers had to be a non-specialist without advanced training in diabetes care.\n\n(3) The nurses should not have undergone post-basic or advanced training in diabetes care.\n\n(4) The participants at the clinic had to be able to interact as a team at their workplace during the six-month SRCC.\n\n(5) The participants were either already managing or interacting with diabetes patients prior to enrolment, or if the participants were not currently managing or interacting with diabetes patients, that opportunity had to be provided to them during the six-month study period of the SRCC.\n\nParticipants from the intervention clinics were enrolled in the programme. Participants from the comparison clinics were then selected so that their clinical experience with diabetes patients matched the participants in the intervention arm as much as possible. Participants from the comparison clinics were offered enrolment in an SRCC class after the completion of the data collection.\n\nThe number of participants in each arm of the research is shown in Table 3.\n\nDespite the fairly small number of participants in each pre and post-test group, the validity of the pre and post-test results can be achieved through a meta-analysis of the data. A statistical treatment of the quantitative data in Solomon’s Four Group Design is possible with a meta-analytical approach15. With this approach, the results of different statistical tests (2x2 ANOVA, repeated measure ANOVA and double tailed t-test) are combined to create statistical power without the need for a large sample. Meta-analysis demonstrates how the results from disparate, independent tests of the same hypotheses may be statistically combined even when the significance tests arise through different statistical techniques16.\n\nThis study utilised both quantitative and qualitative methods to achieve the specific research objectives. The frequency and timing of these methods were applied depending on the research arm as described in Figure 1. A summary of the different data collection methods and purpose is shown in Table 4. All interviewers, investigators and examiners were trained regarding the study procedures prior to the conduct of the research to minimise variability in the method of data collection.\n\nQuantitative data collection. All course participants were subjected to the same core data collection methods that aim to describe and measure the effectiveness of the educational programme. The quantitative study tools are as follows. The majority of data collection instruments are available as Extended data17; however, since the multiple choice questions (MCQs) are still being used to assess doctors and nurses, these are only available on request on a case-by-case basis.\n\nMultiple choice questions assessing knowledge\n\nThe SRCC test of clinical diabetes-related knowledge was developed by a team of endocrinologist and diabetes nurses based on a review of the full course curriculum for medical officers and nurses. The medical officers’ version included 79 MCQs made available in English and the nurses’ version included 67 MCQs and was available in English and Malay language. Since these questions are still being used to assess candidates, they cannot be made openly available.\n\nDiabetes Attitude Scale\n\nThe third version of Diabetes Attitude Scale 3 (DAS-3) is a previously validated general measure of diabetes-related attitudes developed by the University of Michigan Diabetes Research and Training Center18. This method has been shown to be suitable for evaluation of professional education programmes provided that they include the specific topic areas measured by the five DAS-3 sub-scales18, including the attitude of HCPs on the need for special training in education, seriousness of type 2 diabetes, the overall value of tight glucose control in diabetes care, psychosocial impact of diabetes on patients, and attitude toward patient autonomy, which is the case with SRCC.\n\nThe test was administered to participants in the pilot study and their results were evaluated by an independent medical education consultancy firm who evaluated each version of the test for reliability using Chronbach’s α. The resulting α of 0.61 was slightly lower than the gold standard of 0.8, but deemed sufficiently reliable for a non-high stakes examination, particularly given the relatively small sample size of the pilot cohort.\n\nObjective structured clinical examination (OSCE)\n\nAn OSCE is a short circuit of stations at which each participant is examined individually by one or more experienced examiners using real or simulated (actor) patients. The OSCE stations were developed by a team of endocrinologists and diabetes nurses, based on a review of the full course curriculum for medical officers and nurses. Here, medical officers were examined at nine stations and nurses at eight stations. At each station, participants were asked to perform a limited number of clinical tasks within a specified time period (15 minutes) during which two trained examiners used a marking scheme to differentiate good performance from poor performance. The examiners were FMS for the medical officers, and senior diabetes educators for the nurses.\n\nFor each observed competency, each examiner assigned a competency ranking (very poor, poor, acceptable, good, very good). Each participant also received an overall station score for each station (1–<2 = fail, 2–<3 = borderline, 3–<4 = pass, 4–<5 = good, 5 = outstanding) as well as domain scores representing the different measured skills within that station.\n\nQualitative data collection. Additional qualitative investigations were also carried out to better understand how knowledge is being acquired and applied in clinical practice and to create a reliable description of the clinical context into which learnings are being integrated. Qualitative investigations were conducted in Arms 1, 2 and 3 clinics, using the following qualitative tools.\n\nIn-depth interviews with SRCC participants\n\nPre- and post-intervention in-depth interviews were conducted with all participants in Arm 1 clinics (23 in total), in their respective clinics, in order to better understand their history with diabetes related training, typical clinical encounters with people with diabetes and daily clinic life. Interviewed participants were also asked about the process of learning from the various learning platforms and each explored the application of new knowledge and skills to clinical practice. These interviews were audio recorded.\n\nKey informant interviews with FMS\n\nFMSs from the intervention (Arm 1 and 2) and comparison (Arm 3) clinics were interviewed pre- and post-interaction to obtain information on the organisation of clinical services for diabetes patients in the 16 health clinics of Arm 1, 2 and 3, in their respective clinics. Post-intervention FMS interviews for Arm 1 and 2 clinics included themes of their involvement and interactions with the SRCC participants during the intervention period. These interviews were audio recorded.\n\nInterviews with patients receiving care from SRCC participants\n\nShort semi-structured interviews lasting between 10 and 15 minutes with 162 randomly selected patients directly after an observed clinical encounter were conducted in Arm 1 clinics, in the respective clinics. Interview data were used to investigate the patients’ experiences and perceptions of the particular observed clinical interaction. The inclusions of patient interviews provided an important opportunity to triangulate data from the direct observations, the HCP interviews, and the patients' experience of the clinical encounter. These interviews were audio recorded.\n\nDirect clinical observation of participants and clinics\n\nLastly, to investigate the translation of new knowledge into actual clinical practice two days of direct observation of clinical practice of the healthcare professionals from the six clinics participating in Arm 1 (23 in total) were conducted by an experienced clinician of the research team. This researcher assessed the level of diabetes related clinical proficiency demonstrated in each observed clinical consultation. In addition to a narrative assessment of the clinical encounter, each observed session was assigned one of five proficiency levels based on criteria established by the National Institutes of Health Proficiency Scale19.\n\nAn observation-based assessment of the clinical environment was completed and focused on the general clinical environment, interactions between patient and HCPs, and the nature of interactions between health staff working in the clinic.\n\nIn order to manage research data effectively and efficiently as well as to ensure confidentiality, all participating clinics and respondents (participants, FMS and patients) were anonymised and only able to be identified by a sequentially generated ID-number during data collection, follow-up, data processing, analyses and publication. All collected information and data, such as consent forms, background information of respondents, and audio files were stored according to the generated ID-number. Paper records were securely stored in locked file cabinets and also scanned in digital form, while digital records of the files including the audio files were stored and backed-up in a password-protected external thumbdrive and hardisk. All data were only accessed by the principal investigator, research project manager and key research assistants. Data entry and transcriptions were conducted by the research assistants and quality checked by the research project manager and principal investigator.\n\nData were analysed using a convergent parallel design approach, a commonly used mixed-methods analytical approach20,21. The purpose of the convergent design is to “obtain different, but complementary data on the same topic” to best understand the research questions22. This design is used to triangulate the methods by comparing and contrasting quantitative statistical results with qualitative findings for corroboration and validation purposes. It is also used to synthesise complementary quantitative and qualitative results to develop a more complete understanding of a phenomenon20.\n\nIndependent data analysis\n\nPre and posttest examination, DAS and OSCE scores were analysed using both the 2x2 ANOVA, repeated measure ANOVA test and independent t-test according to the Solomon’s Four Group Design15.\n\nThe interviews were transcribed and analysed using thematic content analysis20. The lead investigator coded the transcripts according to recurrent themes. As the main purpose of this analysis was to identify how participation in the SRCC impacted clinical competency and whether or not there are contextual factors that support or hinder the translation of new knowledge into clinical practice, these formed the main a priori domains into which sub-categories can be grouped. However, the analysis also allowed for the inclusion of themes not pre-defined in the template.\n\nData from the observations were in the form of descriptive field notes evaluating various aspects of each clinical encounter. Since observational data were collected pre- and post-intervention, the short descriptions of each observed clinical encounter were assessed for observed similarities and differences using an inductive approach in which many observations were analysed to find more abstract generalisations and to build a picture of the phenomenon being studied (i.e. whether the acquisition of new knowledge about diabetes clinical care was translated into practice).\n\nMerging and triangulation of data\n\nThe observational data were combined with the in-depth interviews and key-informant interviews to understand more thoroughly how participation in the educational intervention facilitated the demonstration of new clinical skills, and to further explore the contextual factors that enabled or inhibited the translation of new knowledge into practice. The data were further explored to identify emerging and recurrent trends within and between qualitative data and to the incidence of themes within and across qualitative data types.\n\nQuantitative and qualitative data were merged using common mixed-methods strategies20. Firstly, content areas that were represented in both data types were identified, compared, contrasted and synthesised as findings. Secondly, differences in one set of results were further examined based on defined dimensions found in the other data set in an attempt to identify and explore data that complements or contradicts the findings from the shared content areas. The exact methods and procedures of the merging process would be dependent on the results from each data collection method.\n\nInterpretation of merged data focused on a discussion of the ways in which the quantitative and qualitative data converged, diverged, related to each other and produced a more complete understanding of whether and how the educational intervention results in changes in diabetes care competence.\n\nPrimary outcomes were changes in diabetes-related knowledge, attitudes, skills, and clinical practice. Patients’ perceptions regarding the quality of care provided were classified as a secondary outcome. Other data included patients' assessment of their chronic disease care and providers' perceptions, attitudes and perceived barriers in care delivery.\n\n\nResearch ethics\n\nThe Medical Research and Ethics Committee (MREC) of the MOH approved the study protocol (reference: NMRR-16-449-29909 (IIR), dated 7 April 2016). Permissions from the Family Health Development Division (FHDD) of the MOH and the respective Health District Offices were also obtained prior to the study. The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice (GCP) requirements.\n\nStudy information sheets were distributed to all course participants and observed patients, and informed consent was obtained from all participants and patients prior to their study enrolment. Informed consent forms were made available in English and the Malay language. Confidentiality of personal information was ensured at all times. Participation of the HCP and patients was voluntary.\n\nSince the study included direct observation of clinical encounters, the possibility of observing sub-standard care existed. If diabetes and/or medical care was witnessed that could lead to acute hospitalisation and/or death, the data collector(s) were instructed to immediately raise this concern to the HCP(s), in order to avoid this risk. For substandard diabetes and/or medical care not likely to lead to acute hospitalisation and/or death, investigators were instructed to avoid intervention, so as not to lead to punitive actions toward the involved HCPs, nor to compromise the HCP-patient relationship.\n\n\nDiscussion\n\nThis study has two key strengths. Firstly, the use of the robust Solomon’s Four Group Design enabled the researchers not only to assess the intervention effect, but also the presence of pretest sensitisation and the interaction effects between intervention and pretest. The Solomon’s Four Group design has not been extensively used in recent studies, partly because it is a complicated design and partly because the statistical analysis is rather complicated. Secondly, the use of mixed methods combining both quantitative and qualitative methodologies provided the researchers with data on intervention effects on knowledge, attitudes, clinical skills and practices, and allowed triangulation of the data.\n\nThere are also inherent limitations in the protocol. Observational data collection methods may have limited validity, including the Hawthorne effect23; regarding the interviews, there may be courtesy bias, where participants provide responses that they think the interviewer would like to hear. These potential biases can be addressed through triangulation of data from the different data collection methods24.\n\nPart of the statistical analysis required the transformation of qualitative data for the clinical practice into quantitative data. While we acknowledge the concerns of the limitations of quantitised qualitative data for statistical measurement, the transformation process will be clearly described and only simple statistical measures for differences employed.\n\n\nData availability\n\nNo data are associated with this article.\n\nFigshare: Data collection tools for Impact evaluation of the Steno REACH Certificate Course in Clinical Diabetes Care for health care providers in Malaysia. https://doi.org/10.6084/m9.figshare.11764146.v217.\n\nThis project contains the following extended data:\n\nOSCE Doctors Stations 1–8 Examiners copy (DOCX). OSCE examination guidelines and scoring rubrics for all OSCE stations (doctors).\n\nOSCE Nurse Stations 1–8 Examiners copy (DOCX). OSCE examination guidelines and scoring rubrics for all OSCE stations (nurses).\n\nPre- and post-intervention interview guide, patients (DOCX).\n\nPre-intervention interview guide, doctors and nurses (DOCX)\n\nPre-intervention interview guide, FMS (DOCX)\n\nPre- and post-intervention observation format, doctors and nurses (DOCX).\n\nPost-intervention interview guide, FMS (DOCX).\n\nPost-intervention interview guide, doctors and nurses (DOCX).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nThe multiple choice questions are currently being used as part of the assessment of doctors and nurses undergoing the Steno REACH Certificate Course in Clinical Diabetes Care organised by MOH Malaysia. We would however be willing to share a copy on a case-by-case basis, if we are assured that the confidentiality of the multiple choice questions is maintained. Any queries can be directed to Dr Feisul Mustapha (email: dr.feisul@moh.gov.my).\n\n\nAcronyms and abbreviations\n\nCME Continuing Medical Education\n\nCPG Clinical Practice Guideline\n\nDAS Diabetes Attitude Scale\n\nFHDD Family Health Development Division\n\nFMS Family Medicine Specialist\n\nGCP Good Clinical Practice\n\nHCP Healthcare Provider\n\nLMIC Low and Middle-income Countries\n\nMCQ Multiple choice questionnaire\n\nMOH Ministry of Health Malaysia\n\nMREC Medical Research and Ethics Committee\n\nNCD Non-Communicable Disease\n\nNDR National Diabetes Registry\n\nNHMS National Health and Morbidity Survey\n\nOSCE Objective Structured Clinical Examination\n\nSRCC Steno REACH Certificate Course in Clinical Diabetes Care\n\nT2D Type 2 Diabetes", "appendix": "Author contributions\n\n\n\nFIM, MC, JA-H and UB-C conceived and contributed to the design of the study. FIM and LSC contributed to the acquisition of data. FIM, KKN and MC each wrote first drafts of parts of the manuscript. All authors critically revised the manuscript for intellectual content, and have read and approved the final manuscript.\n\n\nAcknowledgements\n\nWe would like to thank the Director General of Health, Malaysia for his permission to publish this article.\n\n\nReferences\n\nMOH: National Health and Morbidity Survey 2015 (NHMS 2015). Volume II: Non-Communicable Diseases, Risk Factors & Other Health Problems. 2015; Institute for Public Health. Reference Source\n\nWHO: Global status report on noncommunicable diseases 2014. 2014. Reference Source\n\nMOH: Health Facts. 2015. Reference Source\n\nRamli A, Taher S: Managing Chronic Diseases in the Malaysian Primary Health Care – a Need for Change. Malays Fam Physician. 2008; 3(1): 7–13. PubMed Abstract \| Free Full Text\n\nAli MK, Rabadán-Diehl C, Flanigan J, et al.: Systems and capacity to address noncommunicable diseases in low- and middle-income countries. Sci Transl Med. 2013; 5(181): 181cm4. PubMed Abstract \| Publisher Full Text\n\nSamb B, Desai N, Nishtar S, et al.: Prevention and management of chronic disease: a litmus test for health-systems strengthening in low-income and middle-income countries. Lancet. 2010; 376(9754): 1785–97. PubMed Abstract \| Publisher Full Text\n\nCervero RM, Gaines JK: The impact of CME on physician performance and patient health outcomes: an updated synthesis of systematic reviews. J Contin Educ Health Prof. 2015; 35(2): 131–138. PubMed Abstract \| Publisher Full Text\n\nRenders CM, Valk GD, Griffin SJ, et al.: Interventions to improve the management of diabetes in primary care, outpatient, and community settings: a systematic review. Diabetes Care. 2001; 24(10): 1821–33. PubMed Abstract \| Publisher Full Text\n\nReutens AT, Hutchinson R, Van Binh T, et al.: The GIANT study, a cluster-randomised controlled trial of efficacy of education of doctors about type 2 diabetes mellitus management guidelines in primary care practice. Diabetes Res Clin Pract. 2012; 98(1): 38–45. PubMed Abstract \| Publisher Full Text\n\nShojania KG, Ranji SR, McDonald KM, et al.: Effects of quality improvement strategies for type 2 diabetes on glycemic control: a meta-regression analysis. JAMA. 2006; 296(4): 427–40. PubMed Abstract \| Publisher Full Text\n\nSmith S, Bury G, O'Leary M, et al.: The North Dublin randomized controlled trial of structured diabetes shared care. Fam Pract. 2004; 21(1): 39–45. PubMed Abstract \| Publisher Full Text\n\nKirkman MS, Williams SR, Caffrey HH, et al.: Impact of a program to improve adherence to diabetes guidelines by primary care physicians. Diabetes Care. 2002; 25(11): 1946–51. PubMed Abstract \| Publisher Full Text\n\nCarney T, Helliwell C: Effect of structured postgraduate medical education on the care of patients with diabetes. Br J Gen Pract. 1995; 45(392): 149–51. PubMed Abstract \| Free Full Text\n\nStead JW, Dudbridge SB, Hall MS, et al.: The Exeter Diabetic Project: an acceptable district-wide education programme for general practitioners. Diabet Med. 1991; 8(9): 866–9. PubMed Abstract \| Publisher Full Text\n\nBraver MW, Braver Sanford L: Statistical Treatment of the Solomon Four-Group Design: A Meta-Analytic Approach. Psychol Bull. 1988; 104(1): 150–154. Publisher Full Text\n\nRosenthal R: Combining results of independent studies. Psychol Bull. 1978; 85(1): 185–193. Publisher Full Text\n\nMustapha F, Calopietro M, Nielsen KK, et al.: Data collection tools for Impact evaluation of the Steno REACH Certificate Course in Clinical Diabetes Care for health care providers in Malaysia. figshare. 2020; Dataset. https://www.doi.org/10.6084/m9.figshare.11764146.v2\n\nAnderson RM, Fitzgerald JT, Funnell MM, et al.: The third version of the Diabetes Attitude Scale. Diabetes Care. 1998; 21(9): 1403–1407. PubMed Abstract \| Publisher Full Text\n\nNational Institutes of Health: Competencies Proficiency Scale. 5 February 2017. Reference Source\n\nCreswell JW, Clark VLP: Designing and Conducting Mixed Methods Research. 2nd ed. 2011: Sage Publications, Inc. Reference Source\n\nCollins KMT: Advanced sampling designs in mixed research: Current practices and emerging trends in the social and behavioral sciences. In: SAGE handbook of mixed methods in social and behavioral research., Tashakkori A, Teddlie C, Editors. Sage: Thousand Oaks, CA. 2010; 353–377. Publisher Full Text\n\nMorse JM: Approaches to qualitative-quantitative methodological triangulation. Nurs Res. 1991; 40(2): 120–123. PubMed Abstract \| Publisher Full Text\n\nMonahan T, Fisher JA: Benefits of \"Observer Effects\": Lessons from the Field. Qual Res. 2010; 10(3): 357–376. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJonsen K, Jehn KA: Using triangulation to validate themes in qualitative studies. Qual Res Org. 2009; 4(2): 123–150. Publisher Full Text" }	[ { "id": "61088", "date": "20 Apr 2020", "name": "Prashanth N Srinivas", "expertise": [ "Reviewer Expertise I have experience in the design of health policy and program evaluation especially on capacity-building programs in local health systems at district/regional levels. However", "I do not have sufficient expertise in assessing the adequacy of the sample size for the proposed research method." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this paper. This is a study protocol of a study to assess the effectiveness of an educational intervention to non-specialist doctors and nurses in public facilities in two cities in Malaysia, that provide Diabetes care to improve their knowledge, skills and practice with respect to the treatment of Diabetes. By proposing a mixed-methods study, they also aim to understand the processes through which this might occur and the specific contextual characteristics of the facilities that may facilitate improvements. By using the four-group design, they also aim to address concerns of classical pre-post test comparisons often used in the evaluation of the effectiveness of training/educational interventions.\n\nOverall comments\nPlease revise carefully for grammatical errors. There are several minor grammatical errors.\n\nThe entire article is written in the past tense and appears to indicate the study is now completed. However, I believe that is not the case as this is a study protocol. Please review.\n\nThe current status and clarity on the timelines of the study need to be added.\n\nThe background briefly summarises evidence around the effectiveness of CME. Authors state “Despite the importance of CME, few studies have measured the clinical impact of CME on diabetes in a real-world setting”. While I agree with this assertion, the definition of \"CME\" can be so broad so that the lack of evidence is also to do with the nature of various educational interventions that may not be going under the name CME. Overall, the background oversimplifies the complexity of educational intervention to professionals (doctors, nurses, health workers). The few interventions and reviews that the authors refer to had educational inputs as one among various other inputs within a complex intervention setting (for example Smith et al. 20041). Whereas other programs such as the GIANT study examined an intervention of training on the use of existing guidelines. Arguably, the interventions being cited in support are very diverse and complex and difficult to characterize as \"…few studies have measured the clinical impact of CME\". Indeed many of the studies cited do not use the term CME…..this section requires better synthesis of the literature cited and/or integrating specific systematic review evidence if available.\n\nTwo pilots are mentioned. The rationale behind having two pilots, how the first one shaped the next one and how/what were the overall lessons learned from the pilots that informed the intervention design are not stated and appear to be useful contextual information in the protocol.\n\nThe characterization of this study as impact evaluation, as opposed to being a study on the effectiveness may need some reflection. While these are - to some extent - overlapping and perhaps a matter of semantics in some cases, the objectives appear to be a study on the effectiveness of the intervention rather than an impact evaluation. Impact refers to the ability of the program to meet its ultimate goals of reducing DM burden, whereas the protocol largely sticks to proximate outcomes at the level of the direct participants of the program with some effort to go to patent-level and contextual factors. Suggest to revise this or else provide clearer justification in case impact evaluation is being retained.\n\nObjective 3 mentions the objective to examine the influence of contextual factors at the level of the health system and sociocultural levels. The protocol provides insufficient detail to understand how this is done. For example, which are the health system building blocks that they assess and how? What is clearly established is the factors at the level of health service (and not system). Similarly, insufficient details as to the framework/approaches to be used to assess socio-cultural levels (either in their quantitative component or in their qualitative component). A further minor point under study objectives is their characterization of a program objective/intention as a “hope”. Consider/review.\n\nMinor/optional comments/suggestions\nAbstract\nPlease consider providing numbers of doctors and nurses recruited, word limit permitting\nIntroduction\nThe authors cite WHO: Global status report on noncommunicable diseases 2014 in support of their assertion for the highest prevalence of Diabetes in Malaysia in the Asia-pacific region. On p.244 of the report cited in table titled \"Raised blood glucose (fasting glucose ≥7.0 mmol/l (126 mg/dl) or on medication for raised blood glucose or with a history of diagnosis of diabetes), the Crude adjusted estimates for Malaysia do not support this assertion. Clarify.\n\n“Out-reach” has been put in quotes. Unclear as to the purpose. Does this mean that these are not really functioning or are not actually providing outreach services.\n\nAuthors’ assertion on majority of care for Diabetes occurring in public services should be supported with a reference. In other mixed health systems, evidence often is ambiguous on this.\n\nAuthors state “The Malaysian healthcare delivery system is facing increasing pressure to provide quality care to patients with diabetes.”. Unclear as to pressure is being faced by whom? By communities/patients, funders, politically? Review and sharpen.\n\nMethods\nMethods are well described. Consider adding a section on the study setting to help readers understand the nature of populations that these clinics cater to and perhaps also brief overview of a typical clinic being included in the study in terms of its patient load and other health services characteristics.\n\nAdequacy of sample size for four-group design has been addressed, but this may require further details, especially if test scores and other quantitative variables do not show sufficient variation between groups (which may be the case?)\n\nFurther details of the quantitative analysis in terms of variables to be used and how they will be analysed will be useful.\n\nWhile this is not absolutely essential, the study does not seem to clarify the possible pathways through which they expect the change to occur (where it does). Given the use of qualitative methods and convergent study design, it is useful to include a framework or a theory of change or a program logic that clarifies the possible pathways through which they expect the change to occur. Please treat this as a suggestion and not a comment that needs to be addressed.\n\nExpected outcomes do not report on any outcomes from the qualitative data component.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "5689", "date": "07 Jul 2020", "name": "Feisul Mustapha", "role": "Author Response", "response": "General comment: Thank you for the comments - on most parts, your comments are well accepted. We will await any further reviewers before amending the manuscript. 1. Please revise carefully for grammatical errors. There are several minor grammatical errors. Thank you, we will review and amend accordingly. 2. The entire article is written in the past tense and appears to indicate the study is now completed. However, I believe that is not the case as this is a study protocol. Please review. By the time this article was accepted for publication, the study has been completed. This study was done a few years ago. 3. The current status and clarity on the timelines of the study need to be added. We will include a footnote on this. 4. The background briefly summarises evidence around the effectiveness of CME. Authors state “Despite the importance of CME, few studies have measured the clinical impact of CME on diabetes in a real-world setting”. While I agree with this assertion, the definition of \"CME\" can be so broad so that the lack of evidence is also to do with the nature of various educational interventions that may not be going under the name CME. Overall, the background oversimplifies the complexity of educational intervention to professionals (doctors, nurses, health workers). The few interventions and reviews that the authors refer to had educational inputs as one among various other inputs within a complex intervention setting (for example Smith et al. 20041). Whereas other programs such as the GIANT study examined an intervention of training on the use of existing guidelines. Arguably, the interventions being cited in support are very diverse and complex and difficult to characterize as \"…few studies have measured the clinical impact of CME\". Indeed many of the studies cited do not use the term CME…..this section requires better synthesis of the literature cited and/or integrating specific systematic review evidence if available. This is a good comment, with fair points. Our intervention is a very specific scope/definition of CME, and we hope this will assist others in their future work. We agree that readers would benefit from a more thorough discussion on CMEs 5. Two pilots are mentioned. The rationale behind having two pilots, how the first one shaped the next one and how/what were the overall lessons learned from the pilots that informed the intervention design are not stated and appear to be useful contextual information in the protocol. The first pilot was on a much smaller scale – for proof of concept – that it was doable with current available resources and work environment. The lecturers and facilitators for this pilot were all from Steno Denmark, and training local facilitators was part of the work. The second pilot built on the first pilot (having addressed implementation, mostly logistic issues), to ensure that we were able to manage known issues. This second pilot was run by local lecturers and facilitators, observed and guided by colleagues from Steno Denmark. 6. The characterization of this study as impact evaluation, as opposed to being a study on the effectiveness may need some reflection. While these are - to some extent - overlapping and perhaps a matter of semantics in some cases, the objectives appear to be a study on the effectiveness of the intervention rather than an impact evaluation. Impact refers to the ability of the program to meet its ultimate goals of reducing DM burden, whereas the protocol largely sticks to proximate outcomes at the level of the direct participants of the program with some effort to go to patent-level and contextual factors. Suggest to revise this or else provide clearer justification in case impact evaluation is being retained. Point well taken. Effectiveness would be a better terminology for the title. 7. Objective 3 mentions the objective to examine the influence of contextual factors at the level of the health system and sociocultural levels. The protocol provides insufficient detail to understand how this is done. For example, which are the health system building blocks that they assess and how? What is clearly established is the factors at the level of health service (and not system). Similarly, insufficient details as to the framework/approaches to be used to assess socio-cultural levels (either in their quantitative component or in their qualitative component). A further minor point under study objectives is their characterization of a program objective/intention as a “hope”. Consider/review. Point well taken. We didn’t look at all aspects. We only focused on 2 specific aspects i.e. process of diabetes service delivery at the clinic, and patients' perspective or perception. On the use of the word \"hope\" - will revise to \"... we contend that...\" 8. Abstract - Please consider providing numbers of doctors and nurses recruited, word limit permitting Point well taken, will amend 9. Introduction: The authors cite WHO: Global status report on noncommunicable diseases 2014 in support of their assertion for the highest prevalence of Diabetes in Malaysia in the Asia-pacific region. On p.244 of the report cited in table titled \"Raised blood glucose (fasting glucose ≥7.0 mmol/l (126 mg/dl) or on medication for raised blood glucose or with a history of diagnosis of diabetes), the Crude adjusted estimates for Malaysia do not support this assertion. Clarify. Excluding the Pacific Island nations (in the Asia Pacific region) as stated in the text – Malaysia does have the highest prevalence of diabetes. 10. Introduction - “Out-reach” has been put in quotes. Unclear as to the purpose. Does this mean that these are not really functioning or are not actually providing outreach services. Acknowledge it is a loose term, and we could remove the quotation marks. 11. Introduction - Authors’ assertion on majority of care for Diabetes occurring in public services should be supported with a reference. In other mixed health systems, evidence often is ambiguous on this. Our error of omission. The reference is #1 of the existing reference. Data is obtained from a community-based survey – NHMS 2015 12. Introduction - Authors state “The Malaysian healthcare delivery system is facing increasing pressure to provide quality care to patients with diabetes.”. Unclear as to pressure is being faced by whom? By communities/patients, funders, politically? Review and sharpen. Point well taken. Will review to clarify. The pressure comes from all of the above. 13. Methods - Methods are well described. Consider adding a section on the study setting to help readers understand the nature of populations that these clinics cater to and perhaps also brief overview of a typical clinic being included in the study in terms of its patient load and other health services characteristics. This has actually been written up as a separate paper: Mustapha, F.I., et al., Variations in the Delivery of Primary Diabetes Care in Malaysia: Lessons to Be Learnt and Potential for Improvement. Health Services Research and Managerial Epidemiology, 2020. 7: p. 2333392820918744. 14. Methods - Adequacy of sample size for four-group design has been addressed, but this may require further details, especially if test scores and other quantitative variables do not show sufficient variation between groups (which may be the case?) Point taken, however, we still feel that sufficient detail has been provided. 15. Methods - Further details of the quantitative analysis in terms of variables to be used and how they will be analysed will be useful. Point taken, however for we felt that the level of detail is adequate for a protocol paper, as the analysis will be elaborated further in the results paper. 16. Methods - While this is not absolutely essential, the study does not seem to clarify the possible pathways through which they expect the change to occur (where it does). Given the use of qualitative methods and convergent study design, it is useful to include a framework or a theory of change or a program logic that clarifies the possible pathways through which they expect the change to occur. Please treat this as a suggestion and not a comment that needs to be addressed. Thank you for this suggestion. Has this been a full qualitative study per se, such a framework will be useful and appropriate. We contend that the present paper can study these aspects of the health care system and reach relevant conclusions without such framework. Nevertheless, we do agree with your opinion that it could be useful to guide discussions. 17. Methods - Expected outcomes do not report on any outcomes from the qualitative data component. Is the rationale for, and objectives of, the study clearly described? Point taken. This will be clarified further." } ] }, { "id": "63034", "date": "11 May 2020", "name": "Kamaliah Mohamad Noh", "expertise": [ "Reviewer Expertise Primary health care delivery system" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written paper, easy to comprehend and to follow the logical thoughts of the author. The paper addresses a relevant topic i.e. training of front line staff in resource-constrained public primary health care clinics in providing quality diabetic care. The research question, the objectives, the methodology including the statistical analysis and the discussion are in alignment and the writing style contributes to a smooth flow of logical thought. What is interesting is the use of patient reported experience measure (PREMS) as an outcome measure, not a frequent measure used in this country context.\nThe title reflects the content of the paper as written. However, being a pilot study and the purpose sampling frame of clinics from a region, the sampling methodology would not support claim of representativeness to the whole country. The title should reflect this.\nThe abstract has clear research question and objective and stops at the methodology because this is a study protocol paper.\nThis study was implemented in 2016 and the results of this study has already been published through a poster presentation, as in this case entitled “Pilot implementation of a novel post-graduate medical education program: Steno REACH certificate course in clinical diabetes care – Malaysia” by the same first and second authors of this paper at DOI:https://doi.org/10.1016/S0168-8227(16)31353-5[re-1]; as well as a full paper publication in MEDICAL EDUCATION ONLINE 2019, VOL. 25, 1710330 https://doi.org/10.1080/10872981.2019.1710330 entitled “Impact of continuing medical education for primary healthcare providers in Malaysia on diabetes knowledge, attitudes, skills and clinical practices” by Shiang Cheng Lim, Feisul Idzwan Mustapha, Jens Aagaard-Hansen, Michael Calopietro, Tahir Aris and Ulla Bjerre-Christensen2. While it is agreed that a study protocol paper can fill a knowledge gap, the methodology has been well described in the published paper by Lim, S.C. et al. It is suggested the author demonstrate how this current paper can add value to scientific knowledge by being different from the previous publication by Lim, S.C. et al.\nWhat is unique in this current paper under review is the use of PREMS as a secondary outcome measure. The author may want to review the paper to reflect a focus on the PREMS component of this study which has not been described elsewhere. A relevant reference for the author would be Borg S, Eeg-Olofsson K, Palaszewski B, et al. Patient-reported outcome and experience measures for diabetes: development of scale models, differences between patient groups and relationships with cardiovascular and diabetes complication risk factors, in a combined registry and survey study in Sweden. BMJ Open 2018;9:e025033. doi:10.1136/ bmjopen-2018-0250333.\nThis study has relevance to other countries with similar context on the challenges faced in upscaling the pilot into a nationwide roll out. The results of the analysis of the post intervention interviews of the trainees as well as their clinic leads may lead to discussion points of interest.\n\nIt would be helpful if the author clarifies if the intervention i.e. the training module, is original or is it an adaptation of the Steno REACH education programme implemented in China, India, the Middle East, South East Asia and Latin America. A review of references for the interventions in different country settings would be useful for the discussion.\nThis study is an interesting one, with a potential of relevant lessons for other countries with the same context, trying to improve the quality of care of diabetes challenged with resource constraints. However, for the sake of originality, the authors are suggested to review the focus of the paper.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "5506", "date": "18 May 2020", "name": "Feisul Mustapha", "role": "Author Response", "response": "This is a well written paper, easy to comprehend and to follow the logical thoughts of the author. The paper addresses a relevant topic i.e. training of front line staff in resource-constrained public primary health care clinics in providing quality diabetic care. The research question, the objectives, the methodology including the statistical analysis and the discussion are in alignment and the writing style contributes to a smooth flow of logical thought. What is interesting is the use of patient reported experience measure (PREMS) as an outcome measure, not a frequent measure used in this country context. Thank you. The title reflects the content of the paper as written. However, being a pilot study and the purpose sampling frame of clinics from a region, the sampling methodology would not support the claim of representativeness to the whole country. The title should reflect this. Point well taken, however, this was clearly stated in the paper and we have not made any claim on the representativeness to the whole country. The abstract has clear research question and objective and stops at the methodology because this is a study protocol paper. This study was implemented in 2016 and the results of this study has already been published through a poster presentation, as in this case entitled “Pilot implementation of a novel post-graduate medical education program: Steno REACH certificate course in clinical diabetes care – Malaysia” by the same first and second authors of this paper at DOI:https://doi.org/10.1016/S0168-8227(16)31353-5[re-1]; as well as a full paper publication in MEDICAL EDUCATION ONLINE 2019, VOL. 25, 1710330 https://doi.org/10.1080/10872981.2019.1710330 entitled “Impact of continuing medical education for primary healthcare providers in Malaysia on diabetes knowledge, attitudes, skills and clinical practices” by Shiang Cheng Lim, Feisul Idzwan Mustapha, Jens Aagaard-Hansen, Michael Calopietro, Tahir Aris and Ulla Bjerre-Christensen2. While it is agreed that a study protocol paper can fill a knowledge gap, the methodology has been well described in the published paper by Lim, S.C. et al. It is suggested the author demonstrate how this current paper can add value to scientific knowledge by being different from the previous publication by Lim, S.C. et al. As the reviewer rightly points out, a previous conference abstract as well as a full paper have given some concise outline of the study design. However, in this paper a much more thorough description is provided. We contend that this has merit in its own right as a potential source of inspiration for other MOH’s researchers who would like to conduct a state of the art evaluation of their training activities – something that unfortunately is not always the case. What is unique in this current paper under review is the use of PREMS as a secondary outcome measure. The author may want to review the paper to reflect a focus on the PREMS component of this study which has not been described elsewhere. A relevant reference for the author would be Borg S, Eeg-Olofsson K, Palaszewski B, et al. Patient-reported outcome and experience measures for diabetes: development of scale models, differences between patient groups and relationships with cardiovascular and diabetes complication risk factors, in a combined registry and survey study in Sweden. BMJ Open 2018;9:e025033. doi:10.1136/ bmjopen-2018-0250333. We thank the reviewer for drawing our attention to this reference, which we will use in our future work. This study has relevance to other countries with similar context on the challenges faced in upscaling the pilot into a nationwide roll out. The results of the analysis of the post intervention interviews of the trainees as well as their clinic leads may lead to discussion points of interest. Thank you. We agree that the findings are of relevance outside the Malaysian context, and that a thorough evaluation of post-graduate on the job training provides a sound basis for decision making. It would be helpful if the author clarifies if the intervention i.e. the training module, is original or is it an adaptation of the Steno REACH education programme implemented in China, India, the Middle East, South East Asia and Latin America. A review of references for the interventions in different country settings would be useful for the discussion. Though Steno Diabetes Center Copenhagen has extensive experience from many years of diabetes-related training of health care professionals in all parts of the world, the SRCC was specifically developed for the Malaysian context. From Steno’s perspective, the SRCC is a novel training module that they have never done before. This study is an interesting one, with the potential of relevant lessons for other countries with the same context, trying to improve the quality of care of diabetes challenged with resource constraints. However, for the sake of originality, the authors are suggested to review the focus of the paper. We hope that the comments above have clarified the position of this study protocol. As for the results, another paper is currently on the way that very much takes into account the various issues raised by the reviewer – which we agree are valid concerns." }, { "c_id": "5757", "date": "02 Feb 2021", "name": "Kamaliah Noh", "role": "Reviewer Response", "response": "I confirm that I have read the responses by the author which I have found that these responses have satisfactorily addressed my comments. I am of the opinion that the revised paper is of an acceptable scientific standard." } ] } ]	1	https://f1000research.com/articles/9-98
https://f1000research.com/articles/9-94/v1	07 Feb 20	{ "type": "Case Report", "title": "Case Report: Xanthogranulomatous salpingo-oophoritis associated to endometriosis – are these different histologic expressions of the same disease?", "authors": [ "Ana Portela Carvalho", "Ana Costa Braga", "Hélder Ferreira", "Ana Costa Braga", "Hélder Ferreira" ], "abstract": "Xanthogranulomatous inflammation is characterized by the presence of foamy histiocytes associated with other inflammatory cells like lymphocytes, plasma cells and neutrophils. It is a rare inflammatory process, which has been more frequently described in chronic pyelonephritis and cholecystitis. Xanthogranulomatosis usually triggers a large distortion of the affected organ, which is secondary to the severe inflammatory response that characterizes this type of lesion. Only a few cases of xanthogranulomatous salpingo-oophoritis have been published to date. Here, we report the case of a xanthogranulomatous salpingo-oophoritis in a patient with endometriosis, suffering from chronic pelvic pain and long-standing infertility. The association between endometriosis and xanthogranulomatous inflammation is extremely rare and can possibly represent a severe histologic expression of this common disorder.", "keywords": [ "salpingitis", "endometriosis", "infertility", "xanthogranulomatous salpingo-oophoritis" ], "content": "Introduction\n\nXanthogranulomatous salpingo-oophoritis (XGSO) is an uncommon form of salpingitis, which is associated with a prominent acute and chronic inflammatory infiltrate with admixed foamy histiocytes1–4. The presence of this xanthogranulomatous inflammation has been described in several organs, most commonly in kidney or gallbladder, in association with chronic pyelonephritis or cholecystitis, respectively1. It is, however, an extremely rare finding in pelvic organs.\n\nXGSO has most commonly been associated with pelvic inflammatory disease, but it has also been described in the presence of intrauterine contraceptive devices, extensive endometriosis, ineffective antibiotherapy, abnormalities in lipid metabolism and with the administration of contrast agents1–5. Few cases of XGSO in patients with leiomyomata have also been published1,4,6. Subclinical bacterial infection seems to intervene and several agents have been implicated, such as Actinomyces, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Staphylococcus viridans, Bacteroides fragilis, Candida glabrata and Group B Streptococci1. Non-infectious causes have also been pointed. Nevertheless, the aetiology of XGSO remains unknown.\n\nPatients with XGSO usually present with signs of pelvic inflammatory disease, notably, pelvic pain, fever and abnormal bleeding. Treatment with antibiotics and/or surgery is required and the diagnosis of this condition is only possible after histological examination1,2.\n\nThe differential diagnosis includes: pseudoxanthomatous salpingitis and granulomatous salpingitis. The presence of acute and chronic inflammatory infiltrate differentiates XGSO from pseudoxanthomatous salpingitis, which is characterized by xanthoma cells and pigment without prominent inflammatory component, and from granulomatous salpingitis, where granulomas are present2,3.\n\nHere, we report the case of a XGSO in a patient with chronic pelvic pain and infertility associated with endometriosis.\n\n\nCase report\n\nA 35-year-old woman, with no other relevant previous medical or surgical history, presented with 6 years of primary infertility and severe dysmenorrhea and dyspareunia for nearly a year. No other symptoms were described, like dyschezia, dysuria or abnormal vaginal bleeding. The patient had been previous diagnosed with stage III pelvic endometriosis. This diagnosis was histologically established after two abdominal diagnostic laparoscopies in the context of the infertility evaluation. In the last surgery, a left side salpingectomy and adhesiolysis were performed, with limited post-operative improvement.\n\nThe patient was then referred to our Chronic Pelvic Pain Unit due to clinical worsening. On pelvic examination, a painful area located at the right uterosacral ligament was identified by bimanual exam, without pelvic masses. No other physical abnormalities were detected. A pelvic transvaginal ultrasonography was performed, identifying a large uterus with sonographic signs of adenomyosis. Magnetic resonance imaging showed an hyposignal at T1 and T2 sequences, suggestive of an endometriotic infiltration lesion at the right uterosacral ligament location. This finding correlated with the painful area detected at bimanual exam.\n\nTaking in consideration these clinical and imagiological findings, it was decided to perform a laparoscopy. Several adhesions between the uterus and the anterior rectum were identified. The right Fallopian tube was attached to a 2cm nodule in the right ovarium, which was highly suggestive of severe endometriotic infiltration (Figure 1 and Figure 2). In addition to extensive adhesiolysis, a right salpingectomy and oophorectomy were also performed.\n\nThe right fallopian tube was 3.9cm in length, 0.4 cm in diameter and had a golden yellowish colour. The lumen was dilated, with thickened plicae and wall. The serosal surface was irregular, suggesting focal bilateral adhesions. The ovarian mass consisted of an irregular brown to yellowish nodule of tissue with 2cm of diameter. Both the fallopian tissue and the ovarian nodule were paraffin embedded and haematoxylin-eosin stained slides were examined.\n\nHistopathological examination of the fallopian tube showed abundant infiltration of the lamina propria by foamy histiocytes mixed with some inflammatory cells, including lymphocytes, plasma cells and occasional neutrophils, and there was no intervening stroma, conditioning tightly packing of the fallopian tube plicae. The histiocytes were intimately contiguous to the muscle wall and the subserosa of the fallopian tube and there was serosa fibrosis, with appearance of focal adhesions (Figure 3–Figure 7). The histiocytes appeared to contain abundant lipid material. Red cell extravasation was identified throughout the lesion. A similar finding was present in the ovarian nodule, where this pattern of inflammation was in close relation to normal ovarian tissue. No microorganisms were identified using periodic acid–Schiff, methenamine silver, acid-fast bacilli and Gram stains. Immunohistochemical stain was performed on paraffin-embedded sections and demonstrated strong CD68 staining in foamy histiocytes (Figure 8). No pigments, multinucleated giant cells, granulomas or foci of endometriosis were present in both specimens. The fallopian tube epithelium has reactive aspect, without prolifferative foci. These findings were diagnostic of XGSO.\n\nThis patient had a significant symptomatic improvement after surgical treatment with sustained clinical response. A close follow-up with regular gynaecological appointments was performed, and no symptomatic recurrence, nor surgical adverse outcomes were detected to date.\n\n\nDiscussion\n\nEndometriotic lesions are characterized by the presence of blood and endometrial shedding, representing a favourable trigger for the development of chronic inflammation and fibrosis. Classically, this disorder causes pelvic dysfunction and anatomical distortion that both lead to chronic pelvic pain and infertility. The pathologic finding of xanthogranulomatous inflammation may represent a severe form of endometriotic lesions, which could explain the recurrence of symptoms in this patient.\n\nIdrees et al. described a progressive spectrum of pathologic changes, from pure endometriosis to mixed endometriotic and xanthogranulomatous inflammation, and finally to only XGSO lesions2. The endometrioid implant shedding and the chronic inflammatory process characteristic of endometriosis could explain the xanthomatous process and the accumulation of excessive foamy histiocytes2. In this case, we observed a complete replacement of the endometriotic tissue, which was previously documented in prior surgeries, by foamy histiocytes. The current finding of a destructive xanthogranulomatous inflammatory process, in the absence of endometriotic foci, make us speculate that, probably, endometriosis reached a “burnout phase”, as postulated by other authors2. Moreover, no other predisposing conditions to the development of XGSO were identified.\n\nIn conclusion, a long history of histologically documented endometriosis with multiple previous surgical treatments may lead to the development of a chronic exaggerated inflammatory response, as found in XGSO. A xanthogranulomatous inflammation may represent a rare but aggressive expression of such a common disorder, as is endometriosis.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and images was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nGray Y, Libbey NP: Xanthogranulomatous salpingitis and oophoritis: a case report and review of the literature. Arch Pathol Lab Med. 2001; 125(2): 260–3. PubMed Abstract\n\nIdrees M, Zakashansky K, Kalir T: Xanthogranulomatous salpingitis associated with fallopian tube mucosal endometriosis: a clue to the pathogenesis. Ann Diagn Pathol. 2007; 11(2): 117–21. PubMed Abstract \| Publisher Full Text\n\nFuruya M, Murakami T, Sato O, et al.: Pseudoxanthomatous and xanthogranulomatous salpingitis of the fallopian tube: a report of four cases and a literature review. Int J Gynecol Pathol. 2002; 21(1): 56–9. PubMed Abstract \| Publisher Full Text\n\nHowey JM, Mahe E, Radhi J: Xanthogranulomatous salpingitis associated with a large uterine leiomyoma. Case Rep Med. 2010; 2010: 970805. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLavoué V, Vigneau C, Duros S, et al.: Which Donor for Uterus Transplants: Brain-Dead Donor or Living Donor? A Systematic Review. Transplantation. 2017; 101(2): 267–73. PubMed Abstract \| Publisher Full Text\n\nAbeysundara PK, Padumadasa GS, Tissera WGM, et al.: Xanthogranulomatous salpingitis and oophoritis associated with endometriosis and uterine leiomyoma presenting as intestinal obstruction. J Obstet Gynaecol Res. 2012; 38(8): 1115–7. PubMed Abstract \| Publisher Full Text" }	[ { "id": "83543", "date": "23 Apr 2021", "name": "Jian-Jun Wei", "expertise": [ "Reviewer Expertise gynecologic pathology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report provided a new case of fallopian tube XGSO in a 35 yrs old women. The clinical history, image and histologic findings were well presented. However, the title and major conclusions are not well supported by this case report.\nAuthors concluded that this XGSO was a severe histologic expression of endometriosis based on clinical history. The presented evidences in supporting such connection seems to be lacking or not presented.\n\nXGSO can be related to many different diseases (Int J Gynecol Pathol Sep;39(5):468-472)1. Endometriosis is one of possible cause. Evidence of endometriosis in associated with XGSO by histologic or laparoscopic findings was not shown or documented. The cause of this XGSO may be unknown.\n\nFigures 3-7 are repetitive can be reduced while low power view of configuration of XGSO in fallopian tube should be added.\n\nAuthor speculated a burnout phase of endometriosis for this XGSO. this can be proved by adding CD10 stain to highlight the rim or residual endometrial stromal cells even in a \"Burnout phase\" of endometriosis.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "96889", "date": "08 Nov 2021", "name": "Üzeyir Kalkan", "expertise": [ "Reviewer Expertise endometriosis", "minimally invasive surgery." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report provided a new case of a rare pathology of fallopian tube diagnosed as XGSO. The clinical history, histologic findings and quality of macro and micro images are well documented and presented. Although the title is very attractive, the conclusions do not strongly support this attraction in this case report.\nIn XGSO, usually a clinical history of pyometra or PID exists (Furuya et al. (20021)). In this case, there is no data represented that excludes a history of PID. Also, a clear indication of left salpingectomy which was performed in the last surgery is not reported. Was it due to a pyosalpinx/ hydrosalpinx? The pathologic report of left salpingectomy (if accessible) may add to the case report.\nUsually, pseudoxanthomatous salpingitis is associated with endometriosis (Furuya et al. (20021), Seidman et al. (20152)), and XGSO is rarely associated with endometriosis. In this case, no pigments, multinucleated giant cells, granulomas, or foci of endometriosis were present in the specimens and this mixed inflammatory infiltrate distinguished from the pseudoxanthomatous salpingitis. However, evidence of endometriosis in associated with XGSO by histologic or laparoscopic findings is not shown or documented. The cause of this XGSO may not definitively be claimed that it is due to endometriosis.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-94
https://f1000research.com/articles/8-1444/v1	16 Aug 19	{ "type": "Research Article", "title": "Acute and sub-acute toxicity study of the root extracts of Fagaropsis hildebrandtii in mice and evaluation of their antimicrobial effects", "authors": [ "Beatrice Mwende Muia", "James Mucunu Mbaria", "Laetitia Wakonyu Kanja", "Nduhiu Gitahi", "Paul Onyango Okumu", "Mitchel Otieno Okumu", "James Mucunu Mbaria", "Laetitia Wakonyu Kanja", "Nduhiu Gitahi", "Paul Onyango Okumu", "Mitchel Otieno Okumu" ], "abstract": "Background: Among the Kamba community of Kenya, roots of Fagaropsis hildebrandtii (FH) are boiled and used in managing cough, fertility problems, and microbial infections. The safety of this plant in oral administration and the validity of the ethnomedical claims is unverified. This study evaluated the toxicity of the aqueous and hexane root extracts of FH in mice and antimicrobial effects against Staphylococcus aureus, Salmonella typhimurium and Candida albicans. Methods: Doses (300 and 2000mg/kg) of the extracts were administered orally to mice for 14 days. The weight, feed, and water consumption, organ weight of mice and gross macroscopy of the liver were used in evaluating acute toxicity. Mice were additionally treated with 250, 500, and 1000mg/kg body weight doses of the extracts for 28 days and haematological, biochemical, and histological parameters noted. The minimum inhibitory and minimum bactericidal/fungicidal concentrations (MIC; MBC/MFC) of the extracts against the aforementioned pathogens were determined by broth dilution. Results: Acute oral toxicity of the extracts was >2000mg/kg, there were dose dependent changes in haematological and biochemical parameters, all female mice died when treated with doses of 1000mg/kg and doses ≥500mg/kg caused tubular degeneration and haemorrhage of the kidney, cloudy swelling of hepatocytes, and multifocal necrosis and pyknosis in the liver. The MBC/MIC ratio of each of the extracts against Staph. aureus and S. typhimurium was 2, while C. albicans was not sensitive to any of the extracts. Conclusions: Long term use of root extracts of FH was associated with dose-dependent changes in the kidney and liver of mice and changes in biochemical and haematological parameters. Root extracts of FH are bactericidal against Staph. aureus and S. typhimurium but have no effect on C. albicans. Future work should aim at identifying the metabolites responsible for the observed toxic and bactericidal effects of the roots of FH.", "keywords": [ "Fagaropsis hildebrandtii", "toxicity", "antimicrobial activity", "mice." ], "content": "Introduction\n\nIn the last two decades, the demand for and access to complementary and alternative medicine has grown exponentially1. This has been down to a host of reasons including the integration of complementary and alternative forms of medicine into mainstream healthcare, as exemplified in Taiwan1, a general lack of access and means to afford therapies from the developed world by people in impoverished areas in Africa and Asia, and cultural attitudes towards health in different parts of the world2,3. Notwithstanding, complementary and alternative medicine continues to be sidelined in discussions on the global stage in matters pertaining to public health4. This state of affairs means that there is little impetus to unravel the untapped potential that medicinal species used in complementary and alternative medicine hold. The safety profile of some of the medicinal plants used in complementary and alternative therapies are also unknown despite positive ethnomedical reports emanating from various communities around the world. This therefore calls for a thorough scrutiny of not only the ethnomedical claims but also the safety of the medicinal plants used in such therapies.\n\nCandida albicans, Salmonella typhimurium, and Staphylococcus aureus are some of the most common human pathogens in low and middle-income countries5,6. The development of antimicrobial resistance to these pathogens is an ongoing problem and is of public health relevance7,8. Thus, the need for alternatives to the present drugs indicated for infections by these microorganisms has never been more important.\n\nFagaropsis hildebrandtii (Figure 1) is a deciduous shrub/tree which grows up to a height of 24 meters9. It has nice smelling fruits when unripe, which turn red when they ripen. It is native to Ethiopia, Somalia and Kenya9. Among the Kamba community of Kenya, it is known as “muvindavindi”9. Some ethnomedical claims associated with the plant include treatment of pneumonia, chest pain, arthritis, stomach pains, ulcers, malaria, internal abscesses, epilepsy and resolving infertility in women, as well as chest and respiratory infections9. This is administered as follows: the root/bark is soaked in water or boiled and administered as an infusion or decoction at a dose of one glass three times daily9. Not only is there a dearth of scientific literature supporting these medicinal claims, but also the safety of this plant for various ethnomedical applications may only be assured after extensive scientific scrutiny. This study therefore aimed to determine the safety of root extracts of F. hildebrandtii on oral administration in mice, as well as to evaluate the antimicrobial effects of the extracts against Staph. aureus, S. typhimurium, and C. albicans which are gram positive, gram negative, and fungal microorganisms, respectively. The null hypothesis for this study was that root extracts of F. hildebrandtii are safe on oral administration in mice. Up to 75% of all experiments on toxicity make use of rodents (rats or mice)10 and it is assumed that the effects detected in rodents are the same as those that would be induced in humans unless there is some specific information of species differences in the test response10. Moreover, the response of the test animal is generally considered representative of the average sensitivity of the human population and that for some members of the human population, the risk to health may be much higher10.\n\n(A) Whole plant and (B) aerial parts. Photos by BM.\n\n\nMethods\n\nHPLC grade hexane was purchased from Sigma Aldrich (St. Louis MO, USA). All chemicals used in preparing various reagents for phytochemical screening were analytical grade and high purity. Two bacteria (gram-positive: Staphylococcus aureus ATCC 25923; gram-negative: Salmonella typhimurium ATCC 7222569) and one fungus (Candida albicans ATCC 10231) were provided by the Microbiology section of the Department of Public Health, Pharmacology and Toxicology of the University of Nairobi.\n\nFresh roots of F. hildebrandtii were collected from Kilome constituency, Kitaingo sub location in Makueni County in Kenya (GPS coordinates: 1.7726° S, 37.4498° E). Identification and authentication of the plants were done at the herbarium, Department of Land Resource Management, University of Nairobi. Identification and authentication involved the determination of the botanical origin of the plant, determination of the scientific binomial name, determination of the vernacular name, site of collection, habitat and season of collection, altitude and the parts collected11,12. The keys available in the book titled ‘Kenya trees, shrubs, and lianas’11 was used to arrive at the said genera and the specific epithet.\n\nThe collected roots were gently washed in tap water to remove dirt. They were then shade dried in the laboratory at room temperature for ~2-3 weeks. After completely drying, plant material was pulverized by use of a mechanical mill and subsequently sieved using a 0.45µm pore Whatman® membrane filter to obtain a powder of suitable consistency. The powdered material was weighed and divided into two portions. One portion (~500g) was mixed with ~2000 ml of sterile distilled water, allowed to macerate for 72 hours and centrifuged at 3000 rpm for 10 minutes. The supernatant was collected, filtered through a 0.45 µm membrane filter, and the filtrate lyophilized, and appropriately stored in an amber coloured bottle (henceforth known as AQRFH). To the second portion, 1250 ml of hexane was added and the mixture allowed to macerate for 72 hours, centrifuged at 3000 rpm for 10 minutes, and the supernatant filtered using a 0.45 µm membrane filter. The filtrate was then reduced by use of a rotary evaporator at 40℃ to obtain a dried product (henceforth known as HEXRFH).\n\nAQRFH and HEXRFH were analysed for the presence of various phytochemical metabolites, including alkaloids, cardiac glycosides, flavonoids, phenolics, saponins, phytosterols, tannins and triterpenes using standard procedures13. See Table 1 for the exact tests carried out.\n\n+: Present, -: absent\n\nAdult healthy male and female albino mice (Mus muculus; BALB/c strain; n=66: 42 females, 24 males; age: 8 to 12 weeks; body weight: 20–30 g) were used for this study. The number of animals used were based on the Organization for Economic Corporation and Development (OECD 423) test guidelines14 (Extended data: Table S115). Female mice were nulliparous and not pregnant and all animals were sourced from the Animal House of our institution (University of Nairobi). They were housed in polypropylene cages in the laboratory for at least one week to acclimatize to standard conditions (temperature: 25±3°C; relative humidity: 56–60%; 12 hours of light and 12 hours of darkness). Fluorescent room lights were switched on at 0600h–1800h (light cycle) and off at 1800h–0600h (dark cycle). They were fed on standard mice pellets from a commercial feed supplier (Unga Group Plc, Kenya) and water was provided ad libitum.\n\nAll efforts were made to ameliorate any suffering of the animals by adopting the OECD 423 test guidelines14 as well as the recommendations of the Biosafety, Animal Use and Ethics Committee (BAUEC) of the University of Nairobi (BAUEC/2018/163) (Extended data: Figure S115).\n\nAll treatments on the experimental animals were carried out in rat cages during the daytime/light cycle (0600h to 1800h) at the animal holding unit of the Department of Public Health, Pharmacology, and Toxicology, University of Nairobi.\n\nAcute toxicity study. The acute toxic class (OECD 423) test guidelines on acute oral toxicity were used16. In total 18, 8–12-week-old female albino mice of 18–20 g were labelled from 1–6 in such a way that there were three animals bearing each number17. The number assigned to each animal was then written on a piece of paper (n=18), folded and placed on a receptacle which was then shaken17. A paper was withdrawn at random and the animal that bore the number was assigned into the respective cages labelled 1 (distilled water control group), 2 (extra virgin oil control group), 3 (300mg/kg AQRFH extract group), 4 (2000mg/kg AQFRH group), 5 (300mg/kg HEXFRH group) and 6 (2000mg/kg HEXFRH group)17. Each animal was individually weighed at day 0, 7, and 14 and the observation of the weights noted (Extended data: Table S215). The mice were then fasted for 4 hours before dosing.\n\nTwo doses (300 and 2000 mg/kg of body weight) of either AQRFH or HEXRFH were prepared by accurately weighing the extracts into 250 ml volumetric flasks, mixing well in distilled water or extra virgin oil respectively, and making up to the mark with either distilled water or extra virgin oil. Dosing of mice followed the OECD 423 test guidelines (i.e. 1ml per 100g body weight of mice)14. Therefore, using the weights of mice as a guide, suitable amounts (in ml, and corresponding to the appropriate doses) of either AQFRH or HEXFRH suspended in distilled water and extra virgin oil, respectively, were drawn into 2ml syringes attached to a gastric gavage and administered to mice. Two groups (Groups 1 and 2) served as controls, and mice in these groups received by gastric gavage a daily dose of distilled water and extra virgin oil. Group 3 mice received a 300 mg/kg dose of AQRFH, Group 4 mice received a 2000 mg/kg dose of AQRFH, Group 5 mice received a 300 mg/kg dose of HEXRFH, and Group 6 mice received a 2000 mg/kg dose of HEXRFH.\n\nFood and water consumption of the mice per treatment was evaluated at 0, 7 and 14 days after treatment (Extended data: Table S315). At the end of the 14 days, the mice were sacrificed and organs such as the liver, kidney, lungs and spleen were weighed and used to calculate the relative mean organ weight as below:\n\n\n\nThe data on relative mean organ weight of mice in the acute toxicity protocol is summarized in Extended data: Table S4>15.\n\nSub-acute toxicity studies. This study was carried out using 24 male and 24 female albino mice (20–30g). They were grouped by randomized complete block design into 6 treatment groups and 2 control groups (n=6 mice/group). Control group mice received an oral daily dose of 0.6ml distilled water (aqueous extract control) and 0.3ml of extra virgin oil (hexane extract control) for 28 days. Treatment group mice received graded doses (250, 500 and 1000mg/kg body weight) of AQRFH and HEXRFH which were administered once daily for 28 days. The body weights of the mice were noted at day 0, 7, 14,21, and 28 and the doses adjusted accordingly (Extended data: Table S515).\n\nClinical observations. Changes in fur and skin colour, mucous and eye membranes, respiratory, circulatory autonomic and central nervous systems were noted16. Other observations to be noted included tremors, convulsions, confusion, salivation, diarrhoea, coma and death. Observations were done at intervals of 30 minutes, 4 hours, 24 hours, 48 hours, 7 days, 14 days, 21 days and 28 days16.\n\nBody weight. Individual body weights of mice in the sub-acute toxicity protocol were recorded on day 0, 7, 14 and 28 (Extended data: Table S515). Body weight was also recorded prior to the first dosing on day 0 and terminally (after fasting) prior to necropsy (Extended data: Table S515). The food and water consumption in the sub-acute toxicity protocol are summarized in Extended data: Table S615.\n\nNecropsy and organ weight. All animals were fasted overnight prior to necropsy. The animals were euthanized in 0.5% v/v halothane (Piramal Enterprises, India; Batch number K37L17B) in a bell jar and vital organs (stomach, kidney, lung, spleen, heart and liver) were excised from both male and female mice, washed gently and observed macroscopically for lesions or any abnormal signs. Organ weights (absolute and relative) were determined for the excised organs by placing them in absorbent paper for a few minutes and weighed. The relative organ weight of each mice was then determined as below:\n\n\n\nThe relative mean organ weight of mice in the sub-acute toxicity protocol are summarized in Extended data: Table S715.\n\nClinical pathology. At the end of dosing, mice were fasted overnight prior to scheduled necropsy. On day 29, mice were anaesthetized using halothane inhalation in a bell jar, blood samples were collected by use of cardiac puncture into vacutainers containing anticoagulant (EDTA for haematological parameters) and without anticoagulant (plain tubes for biochemical parameters). About 0.5 ml of the blood in plain tubes was centrifuged at 3000 rpm for 10 minutes to obtain serum, which was then stored at −20 °C awaiting further use in biochemical assay of aspartate amino transferase (AST), alanine amino transferase (ALT), total protein (TP), urea and creatinine using specific kits (Catalyst AST Aspartate amino transferase-98-11067-01, Catalyst ALT/GPT Alanine amino transferase-98-11069-01, Catalyst TP Total Protein-98-11085-01, Catalyst BUN Urea-98-11070-01, and Catalyst CREA Creatinine-98-11074-01: Magnum Veterinaria, Laagri Arimaja, Vae 16, Estonia). In addition, ~1.3ml of the blood collected in the anticoagulant containing vacutainers was used to evaluate levels of haematocrit (HCT), total white blood cell (WBC) count, total red blood cell (RBC) count, haemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC) using IDEXX kits (One Idexx drive, Westbrook, Maine, 04092, United States).\n\nHistopathological studies. Histopathological studies were carried out on organ samples of liver and kidney of the mice. These organs were surgically removed from the mice and fixed in 5% buffered formalin. Tissue samples were then dehydrated in graded doses of ethanol (70–99.9%; Fischer Scientific Ltd.; Batch number 0556512, washed in toluene (Fischer Scientific: Lot no: 193377, and enclosed in paraffin (Honeywell FlukaTM: 18634H). Thin tissue sections of 5µm were obtained on a rotatory microtome and stained with haematoxylin-eosin dye and analysed microscopically for any pathological alterations. The photomicrographs obtained were labelled for identification.\n\nAntimicrobial activity. Staph. aureus (gram positive bacteria), S. typhimurium (gram negative bacteria) and C. albicans (fungus) were cultured on blood agar (Oxoid Ltd.; Lot number 1623360) and incubated overnight at 37°C in an incubator (Memmert, Germany). The cultures were suspended in 5 ml sterile physiological buffed saline and turbidity adjusted to 0.5 McFarland to give a concentration of approximately 1.5 × 108 colony forming units per ml (CFU/ml). In total, 800 mg of AQRFH and HEXRFH were dissolved in 3ml of sterile distilled water and virgin oil, respectively. Serial dilutions ranging from 3.125–400 mg/ml of the extracts were then prepared using sterile peptone water (Oxoid Ltd; Lot number 1721983). Subsequently, using a sterile 1ml pipette, the adjusted individual micro-organisms (Staph. aureus, S. typhimurium and C. albicans were inoculated into every tube of the diluted plant extracts to give a final concentration of 5×106 CFU/ml. The tubes were then incubated at 37°C for 24 hours. Serial dilutions of the plant extracts devoid of micro-organisms were used as negative control. Positive control tubes had the respective microorganisms without serial dilutions of the plant extracts. Peptone water was used to blank the spectrophotometer. Flucloxacillin (10mg/ml; Dawa Pharmaceuticals Ltd-Kenya; Batch number MB18085 and fluconazole (10mg/ml); Universal Corporation Ltd-Kenya; Batch number 5805073) were used as positive reference compounds of antimicrobial activity.\n\nBaseline optical density at a wavelength of 450 ηm readings for each of the tubes were taken immediately after all tubes had been prepared using a spectrophotometer (Spectronic 21D, Milton Roy, USA). Optical density readings were also taken after 24 hours. A comparison between baseline optical density readings and optical density readings after 24 hours of inoculation of the tubes was made. This was done to determine whether there was growth or not. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of the extracts that inhibited any visible growth of bacteria as determined by the absence of change in the optical density readings between baseline values and those obtained after incubation of the tubes. For the determination of the minimum bactericidal/fungicidal concentration (MBC/MFC), 100µl of broth was taken from all the MIC tubes after 24 hours of incubation at 37°C and sub-cultured aseptically to Plate Count Agar (Oxoid Ltd.; Lot number 1626187). The plates (WOM-CHINA; Lot number RT20171103) were incubated at 37°C for 24 hours and checked for bacterial growth. The lowest concentration of the plant extracts that showed no bacterial growth was considered the MBC/MFC, defined as the lowest concentration of the extract which is responsible for killing greater than 99.9% of the initial bacteria inoculums.\n\nResults of haematological and biochemical tests were expressed as mean ± of standard deviation of three independent observations and analysed using ONE-WAY ANOVA on Statistical Package for the Social Sciences (SPSS, version 21.0). Tukey’s test was used as post hoc. p< 0.05 was considered significant.\n\n\nResults\n\nAQRFH and HEXRFH yielded about 9% and 2.74% of extract, respectively. Preliminary phytochemical screening of AQRFH revealed the presence of alkaloids, flavonoids, phenolics, saponins, steroids, tannins and terpenoids while the screening of HEXRFH revealed the presence of alkaloids, cardiac glycosides, phenolics, saponins, steroids, tannins and terpenoids (Table 1).\n\nAcute toxicity studies were carried out for 14 days as per the OECD 423 guidelines14. No mortality of mice was observed within the first 4 hours of continuous observation, nor after 24 hours. There was also no lethal effect observed after the administration of the extracts for the experimental period of 14 days. Morphological characteristics such as the fur, and skin colour appeared normal. There was no salivation, diarrhoea, lethargy, unusual behaviour or altered respiration. Mean body weight (Extended data: Table S215), food and water intake (Extended data: Table S315), and relative mean organ weight (Extended data: Table S415) of treatment group animals were not significantly different from the control group animals. Figure 2 illustrates the changes in the mean body weight of mice receiving different treatments over the 14-day study period for acute oral toxicity. Figure 3 illustrates the feed and water consumption of the experimental mice over the 14-day study period.\n\nDW: distilled water, EVO: Extra virgin oil, AQ: aqueous, HEX: Hexane, AFC: average food consumption, AWC: average water consumption\n\nOn gross macroscopic examination, there was no necrosis, inflammation or change in size of the lungs, liver, spleen, kidney or heart in mice (Figure 4).\n\nA. Gross macroscopy of the lungs, liver, spleen, kidney and heart excised from a control group mouse, B. Gross macroscopy of spleen, lung, and spleen excised from a mouse treated with a 2000mg/kg dose of AQFRH.\n\nMean body weight of mice that received AQFRH over 28 days. There was no significant difference in the mean weight of control mice relative to the mice treated with 250 or 500mg/kg doses of AQFRH. However, there was a significant difference in the mean weight of control mice relative to the mice (female) treated with a 1000mg/kg dose of AQFRH after 28 days (Extended data: Table S515).\n\nFeed and water consumption in mice treated with graded doses of AQFRH and HEXFRH. Feeding mice on graded doses of the root extracts of F. hildebrandtii over a 28-day period had no significant effect on the feed and water consumption of mice in treatment groups relative to the control (Extended data: Table S615).\n\nRelative mean weight of mice treated with graded doses of AQFRH and HEXFRH. Doses of 250mg/kg and 500mg/kg of AQFRH did not produce any significant effect on the mean weight of mice relative to the controls (Extended data: Table S715).\n\nNone of the doses of HEXFRH had any significant effect on the mean weight of the treatment mice relative to the controls (Extended data: Table S815).\n\nRelative-organ weight ratios of mice treated with AQFRH and HEXFRH. There was no significant change in the mean relative weight of the stomach, left and right kidneys, lung, spleen, heart and liver in mice treated with a 28-day oral dose of 250mg/kg and 500mg/kg of AQRFH, respectively (Extended data: Table S915). A 1000mg/kg dose of AQRFH significantly lowered the mean relative weight of the spleen in both male and female mice (Extended data: Table S915). This dose also significantly lowered the mean relative weight of the liver in male mice, and the stomach and lung in female mice relative to the control group (Extended data: Table S915).\n\nThere was no significant change in the mean relative organ weight of the stomach, left and right kidneys, lung, spleen, heart and liver in mice treated with a 28-day oral dose of 250mg/kg and 500mg/kg of HEXRFH (Extended data: Table S1015). A 1000mg/kg dose of HEXRFH significantly increased the mean relative organ weight of the liver in male mice relative to the control. This dose also produced a significant decrease in the mean relative organ weight of the lungs, spleen and heart in male and female mice, and the stomach of female mice relative to the control group (Extended data: Table S1015).\n\nEvaluation of haematological parameters. A 250 mg/kg dose of AQRFH significantly lowered the mean RBC levels in male mice, while significantly raising the mean platelet levels in both male and female mice relative to the control group (Table 2). This dose also non-significantly increased the mean levels of Hb, HCT, MCV, MCH, and the MCHC in both male and female mice as well as mean levels of WBC in male mice relative to the control (Table 2). This dose also non-significantly lowered the mean RBC levels in female mice relative to the controls. A 500mg/kg dose of AQRFH significantly lowered the mean RBC levels in male mice while significantly raising the mean Hb levels in female mice and the mean platelet levels in male mice relative to the control (Table 2). This dose also non-significantly increased the mean levels of MCHC, MCH and WBC in both male and female mice, and the mean levels of HCT, platelets and MCV in female mice, as well as the mean Hb levels in male mice relative to the controls. A 1000mg/kg dose of AQRFH resulted in the death of all female mice tested (n=6). Thus, no haematological parameter was evaluated for these animals. On the other hand, this dose of extract significantly lowered the mean levels of all haematological parameters evaluated in male mice relative to the controls (Table 2).\n\nValues expressed as mean ± SD (n=3). Hb: Haemoglobin, MCV: Mean corpuscular volume, MCH: Mean corpuscular haemoglobin, MCHC: Mean corpuscular haemoglobin concentration, WBC: White blood cells\n\nA 250mg/kg dose of HEXREF significantly lowered the mean RBC and platelet levels in male and female mice relative to the controls (Table 3). This dose also non-significantly lowered the mean haematocrit, WBC and MCHC levels in both male and female mice, as well as the mean Hb, and MCV levels in female mice relative to the control group. This dose also non-significantly raised the mean Hb and MCV levels in male mice, as well as the MCH levels in both male and female mice relative to the control group. A 500mg/kg dose of HEXRFH significantly lowered the mean RBC levels in both male and female mice, and the mean platelet levels in female mice relative to the control group (Table 3). This dose non-significantly raised the mean Hb, MCHC and WBC levels in male and female mice, as well as the mean levels of Hb, MCH and WBC in male mice relative to the control group. This dose also non-significantly lowered the mean Hb, MCHC and WBC levels in both male and female mice, as well as the MCH levels in male mice relative to the control group. A 1000mg/kg dose of HEXRFH resulted in the death of all female mice tested (n=6). Thus, no haematological parameter was evaluated for these animals. On the other hand, this dose of extract significantly lowered the mean levels of all haematological parameters evaluated in male relative to the controls (Table 3).\n\nValues expressed as mean ± SD (n=3). Hb: Haemoglobin, MCV: Mean corpuscular volume, MCH: Mean corpuscular haemoglobin, MCHC: Mean corpuscular haemoglobin concentration, WBC: White blood cells\n\nEvaluation of biochemical parameters. A 250 mg/kg dose of AQRFH did not produce any significant change in the mean levels of any of the biochemical parameters evaluated (Table 4). This dose produced a non-significant decrease in the mean levels of urea, creatinine and ALT in both male and female mice and AST and TP in male mice relative to the control group. This dose also produced a non-significant increase in the mean levels of AST and TP in female mice relative to the control group. A 500 mg/kg dose of AQRFH significantly raised the mean levels of ALT in female mice relative to the controls (Table 4). This dose produced a non-significant decrease in the mean levels of AST in male and female mice, and in mean levels of ALT, urea, creatinine, and TP in male mice only relative to the controls. This dose also produced a non-significant increase in the mean levels of urea, creatinine and TP in female mice only relative to the controls. A 1000mg/kg dose of the aqueous extract of F. hildebrandtii resulted in the death of all female mice tested (n=6) (Table 4). Thus, no biochemical parameter was evaluated for these animals. On the other hand, this dose of extract significantly raised the mean levels of all biochemical parameters evaluated in male mice relative to the controls.\n\nValues are expressed as mean ± SD of three animals. ALT: Alanine aminotransferase, AST: Aspartate amino transferase, TP: Total protein\n\nA 250 mg/kg dose of HEXRFH produced a significant increase in the mean levels of ALT in female mice (Table 5). This dose produced a non-significant decrease in the mean levels of urea in male and female mice, creatinine in female mice and AST in male mice relative to the controls. This dose also produced a non-significant increase in the mean levels of TP in male and female mice, creatinine and ALT in males, and AST in female mice relative to the controls. A 500 mg/kg dose of HEXRFH significantly raised the mean levels of ALT in both male and female mice relative to the controls (Table 5). This dose produced a non-significant decrease in the mean levels of urea and AST in male mice relative to the controls, and in mean levels of ALT, urea, creatinine, and TP in male mice only relative to the controls. This dose also produced a non-significant increase in the mean levels of urea, creatinine and TP in female mice only relative to the controls. A 1000mg/kg dose of HEXRFH resulted in the death of all female mice tested (n=6) (Table 5). Thus, no biochemical parameters were evaluated for these animals. On the other hand, this dose of extract significantly raised the mean levels of all biochemical parameters evaluated in male mice relative to the controls.\n\nValues are expressed as mean ± SD of three animals. ALT: Alanine aminotransferase, AST: Aspartate amino transferase, TP: Total protein\n\nHistopathological studies on kidney and liver sections of mice receiving AQFRH and HEXFRH over 28 days. In Figure 5, kidney sections A, B, C were characterized by regular glomeruli and normal renal tubules with complete smooth epithelia and clear lumen (X400). Kidney section D (×400) was characterized by degeneration of renal tubules and loss of epithelium. Kidney section E (×400) was characterized by mild tubular degeneration. Kidney section F (×400) was characterized by multifocal tubular degeneration with loss of epithelium. Kidney section G (×400) was characterized by degeneration of tubules and haemorrhage.\n\nA. Kidney section of a mouse treated with a 250mg/kg dose of AQFRH (×400); regular glomeruli (G) and normal renal tubules (T) with complete smooth epithelia and clear lumen (×400), B: Kidney section of a mouse treated with a 500mg/kg dose of AQFRH (×400); degeneration of renal tubule (DT)/loss of epithelium, C: Kidney section of a mouse treated with a 1000mg/kg dose of AQFRH (×400;multifocal tubular degeneration (DT), most of the epithelium is lost , D: Kidney section of a mouse treated with a 500mg/kg dose of HEXFRH (×400); mild tubular degeneration (DT).\n\nIn Figure 6, liver sections A, B, C (×400) were characterized by normal parenchymal architecture with normal hepatic cells that were evenly distributed and separated by sinusoids. Liver sections D and E (×400) were characterized by diffuse cloudy swelling of the hepatocytes indicative of early necrosis with pyknotic nucleus. Liver section F (×400) was characterized by multifocal hepatocyte necrosis with hepatocytes undergoing cloudy swelling with nuclear pyknosis. Liver section G (×400) was characterized by hepatocyte necrosis and pyknosis, cloudy swelling, necrotic hepatocyte and regenerating hepatocyte around the necrotic hepatocyte.\n\na. Liver section of a mouse treated with distilled water (×400), C: congestion, CV: portal vein, P: portal area, b. liver section of a mouse treated with extra virgin oil only (×400); normal parenchymal architecture with normal hepatic cells (H) that are evenly distributed and separated by hepatic sinusoids (S), c. liver section of a mouse treated with a 500mg/kg dose of AQFRH (×400); diffuse cloudy swelling (CS) of the hepatocytes (early necrosis), d. liver section of a mouse treated with a 1000mg/kg dose of AQFRH (×400): multifocal hepatocyte necrosis, CS: cloudy swelling with nuclear pyknosis, N: hepatocyte necrosis, e. liver section of a mouse treated with a 250mg/kg dose of HEXFRH (×400); focal area of hepatocyte degeneration characterized by cloudy swelling, f. liver section of a mouse treated with a 1000mg/kg dose of HEXFRH (×400); hepatocyte necrosis and pyknosis, CS: cloudy swelling/pyknosis, R: regenerating hepatocyte.\n\nMinimum inhibitory concentration of root extracts of F. hildebrandtii against Staph. aureus, Salmonella typhimurium and C. albicans. C. albicans was not sensitive to any of the concentrations of AQRFH and HEXRFH. Staph. aureus was sensitive to a concentration of 100 mg/ml, 200mg/ml and 400 mg/ml of both AQRFH and HEXRFH. Salmonella typhimurium was sensitive to concentrations of 200 mg/ml and 400mg/ml of AQRFH and a concentration of 100mg/ml, 200mg/ml and 400mg/ml of HEXRFH (Table 6).\n\nMinimum bactericidal/fungicidal concentration (MBC/MFC) of the root extracts of F. hildebrandtii against S. aureus, S. typhimurium and C. albicans. C. albicans was not sensitive to any of the concentrations of either AQRFH or HEXRFH. Staph. aureus was sensitive to a concentration of 200mg/ml and 400 mg/ml of both AQRFH or HEXRFH. Salmonella typhimurium was sensitive to a concentration of 400mg/ml only of the AQRFH and a concentration of 200mg/ml and 400mg/ml of HEXRFH (Table 7). Based on the findings of MBC and MIC of the extracts against the tested pathogens, the MBC/MIC ratio of the extracts was found to be 400/200=2; for AQFRH against Salmonella typhi), 200/100=2; for HEXFRH against Salmonella typhi), 200/100=2; for AQFRH against Staph. aureus), 200/100=2; for HEXFRH against Staph. aureus)\n\n\nDiscussion\n\nHerbal medicine is a popular form of medicine but is limited by safety concerns. We believe that the present study provides the first account of the safety of any part of F. hildebrandtii on oral administration in any animal model. Moreover, there has been no report of the secondary metabolites that may be present in the plant. However, it is worth noting that Yiaile and colleagues18 have previously reported on the preliminary composition of Fagaropsis angolensis, a plant of the same genus as our plant of interest. Previous studies have reported that flavonoids, phenolics, tannins, and terpenoids have antimicrobial properties with wide-ranging mechanisms of action19–22. In our case, however, the absence of flavonoids in the hexane root extract of F. hildebrandtii does not appear to have resulted in abolishment of the antimicrobial properties of the extract. This observation may suggest that other phytochemicals distinct from flavonoids may be responsible for the observed antimicrobial properties in the particular extract.\n\nSubstances that possess antimicrobial properties are considered as bacteriostatic agents when the ratio MBC/MIC >4 and bactericidal agents when the same ratio is ≤423. Using this criterion, we suggest that both of our extracts were bactericidal in nature. However, it is our opinion that caution should be exercised in interpreting these results. This is because according to Martins and colleagues24, the higher the MIC values, the more likely that test results evaluated lose clinical relevance. Moreover, other studies have reported much lower MIC values for Tarenaya spinosa24, Buchenavia tetraphylla25, Thymus vulgaris26 and Origanum vulgare26 against Staph. aureus and S. typhi. We posit that the differences in MIC values observed between our findings and those of the aforementioned studies may have something to do with the relative abundance of the phytochemicals in our plant of interest.\n\nChanges in the mean body weight, relative organ weight, and feed and water consumption may be indicative of acute toxicity27–29. In our study, administration of a single dose of aqueous and hexane extracts of F. hildebrandtii did not produce any significant changes in these parameters. Moreover, no animals died during the 14-day study period. It may therefore be inferred that these extracts may not have had any untoward effects in mice when administered orally for 14 days. However, sub-acute treatment resulted in an increase in the weight of female mice. Changes (positive or negative) in the body weight of experimental animals may be indicative of alterations in the physiology of the animals and may stem from the variations in the levels of hormones, disorders of major organs, such as the liver or kidney, or decreased absorption of nutrients as a result of treatment with test substances30. Moreover, sub-acute treatment of mice with a 1000mg/kg dose of the root extracts of F. hildebrandtii was associated with a significant decrease in the mean relative organ weight of the stomach and heart in female mice, liver in male mice, and spleen in both male and female mice. According to Teo and colleagues31, the reduction in body weight of experimental animals may negatively impact the weight of internal organs. Given that the acute and sub-acute treatment of mice with root extracts of F. hildebrandtii did not significantly change the consumption of food and water, we believe that the observed effects on animal weight and internal organs were the result of physiological changes in the model adopted and not a consequence of lower food consumption.\n\nPhysiological/pathological response to toxic insult in man or animals can be gauged by evaluating the haematopoietic system32. In this study, the main significant changes were the dose dependent decrease in RBCs in both male and female mice, and significant decreases in the mean levels of Hb, HCT, MCV, MCH, MCHC, platelets and WBCs at 1000mg/kg in both male and female mice. The observations of these effects following the oral administration of root extracts of F. hildebrandtii may be related to the toxicity caused by the extracts. Detoxification is a key function of the liver. Thus, the accumulation of toxic compounds in the liver is bound to be associated with liver damage. This damage is usually assessed by the determination of serum transaminases (ALT and AST) as well as measurement of total proteins32,33. Significant increases in the serum levels of these enzymes particularly at a dose of 1000mg/kg for both aqueous and hexane extracts of F. hildebrandtii and the observations of cloudy swelling, necrosis, and pyknosis on histological analysis of the liver further confirms that the oral administration of these extracts in mice may indeed cause liver damage at high doses. The kidney is another organ that is considered a frequent target of toxicity32,34. In our study, renal function was evaluated by serum levels of urea and creatinine and by histological analysis. The kidney of mice treated with root extracts of F. hildebrandtii at doses of 500mg/kg and above were characterized by degeneration of renal tubules, loss of epithelium and haemorrhage. Moreover, there were significant increases in the mean levels of urea and creatinine in male mice treated with a 1000mg/kg dose of the aqueous and hexane root extracts of F. hildebrandtii. According to Burtis and Bruns, there is usually an increase in the levels of creatinine when the cortex and/or glomeruli is damaged35; therefore such an increase may be a good indicator of chronic kidney disease36. The histological evaluation of this organ further confirms the damage particularly for mice treated with doses of root extracts of F. hildebrandtii above 500mg/kg.\n\nFinally, mortality is usually an important index of the safety or toxicity of a xenobiotic. Although 14-day acute oral exposure of mice to root extracts of F. hildebrandtii did not result in the death of any of the mice, it is damning that female mice treated with a 1000mg/kg dose of the root extracts of F. hildebrandtii in the sub-acute protocol all died before the end of the experiment, and their male counterparts exhibited significant alterations in biochemical, haematological and pathological indices. According to guidelines by the Organization for Economic Development (OECD), in the event that a xenobiotic causes toxicity in rodent models, females are more susceptible to toxicity than males16,37.\n\n\nConclusion\n\nIn conclusion, the short-term use of the root extracts of F. hildebrandtii at doses of 300 and 2000 mg/kg may not result in toxic manifestations in mice. However, long term administration of the extracts (beyond 14 days) was associated with dose-dependent histopathological changes in the liver and kidney of mice, as well as alterations in haematological and biochemical parameters. Considering that the roots of this plant have various ethnomedicinal indications, including the treatment of pneumonia, arthritis, chest and stomach pain, ulcers, malaria, internal abscess, epilepsy, infertility in women and chest and respiratory infections, our results may be useful as a valuable piece of information on the safety profile of roots of this plant and may be important as a reference material for any future work on F. hildebrandtii. Further evaluation on the potential of this plant to induce carcinogenicity, mutagenicity and genotoxicity is warranted in order to paint a better picture on the complete safety profile of this plant. Studies on the identity of the metabolites responsible for the observed toxicity is also warranted.\n\n\nData availability\n\nFigshare: Underlying data on the study titled 'Acute and sub-acute toxicity study of the root extracts of Fagaropsis hildebrandtii in mice and evaluation of their antimicrobial effects', https://doi.org/10.6084/m9.figshare.9104894.v138; https://doi.org/10.6084/m9.figshare.9101231.v239; https://doi.org/10.6084/m9.figshare.9104480.v340\n\nThis project contains the following underlying data:\n\n- Data on the mean body weight of mice used in the acute toxicity protocol39\n\n- Data on the feed and water consumption in mice in the acute toxicity protocol39\n\n- Data on the relative mean organ weight of mice in the acute toxicity protocol39\n\n- Data on the mean body weight of mice treated with sub-acute toxicity protocol40\n\n- Data on the feed and water consumption in mice in the sub-acute toxicity protocol40\n\n- Data on the relative mean organ weight of mice in the sub-acute toxicity protocol40\n\n- Data on the haematological parameters in mice in the sub-acute toxicity protocol38\n\n- Data on the biochemical parameters in mice in the sub-acute toxicity protocol38\n\nFigshare: Photomicrographs of kidney and liver sections of mice treated with distilled water, extra virgin oil, and graded doses of aqueous and hexane root extracts of Fagaropsis hildebrandtii, https://doi.org/10.6084/m9.figshare.9199406.v215\n\nFigshare: Extended data on the study titled 'Acute and sub-acute toxicity study of the root extracts of Fagaropsis hildebrandtii in mice and evaluation of their antimicrobial effects', https://doi.org/10.6084/m9.figshare.9083348.v141.\n\nThis project contains the following extended data:\n\n- Figure S1: The biosafety, animal use and ethics committee approval document for the study\n\n- Table S1: A summary of the animals used in the study\n\n- Table S2: Effect of distilled water, extra virgin oil, and the aqueous and hexane root extracts of F. hildebrandtii on the mean body weight in mice over a 14-day period\n\n- Table S3: Effect of distilled water, extra virgin oil and the aqueous and hexane root extracts of F. hildebrandtii on feed and water consumption in mice over a 14-day period\n\n- Table S4: Effect of distilled water, extra virgin oil and the aqueous and hexane root extracts of F. hildebrandtii on the relative mean organ weight in mice over a 14-day period\n\n- Table S5: Effect of graded doses of the aqueous root extract of F. hildebrandtii on the mean body weight in mice over a 28-day period\n\n- Table S6: Effect of graded doses of root extracts of F. hildebrandtii on water and feed consumption in mice over a 28-day period\n\n- Table S7: Effect of graded doses of the aqueous root extract of F. hildebrandtii on the mean body weight of mice over a 28-day period\n\n- Table S8: Effect of graded doses of the hexane root extract of F. hildebrandtii on the mean relative organ weight in mice over a 28-day period\n\n- Table S9: Effect of graded doses of the aqueous root extract of F. hildebrandtii on the relative mean organ weight in mice over a 28-day period\n\n- Table S10: Effect of graded doses of the hexane root extract of F. hildebrandtii on the relative mean organ weight in mice over a 28-day period\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors would like to acknowledge the support accorded to them by Dr. Ojoo Rodi, immediate former director of the biosafety, animal care and use committee of the University of Nairobi, and the team of laboratory technologists, including Ms Lucy Mwangi, Mr Joseph Nderitu, Mr Ken Maloba and Mr James Macharia, for their assistance and guidance in the laboratory work. The authors are also grateful to the staff at the animal house, department of public health, pharmacology and toxicology for their support.\n\n\nReferences\n\nHorneber M, Bueschel G, Dennert G, et al.: How many cancer patients use complementary and alternative medicine: a systematic review and metaanalysis. Integr Cancer Ther. 2012; 11(3): 187–203. PubMed Abstract \| Publisher Full Text\n\nLiao YH, Li CI, Lin CC, et al.: Traditional Chinese medicine as adjunctive therapy improves the long-term survival of lung cancer patients. J Cancer Res Clin Oncol. 2017; 143(12): 2425–35. PubMed Abstract \| Publisher Full Text\n\nAliyu UM, Awosan KJ, Oche MO, et al.: Prevalence and correlates of complementary and alternative medicine use among cancer patients in usmanu danfodiyo university teaching hospital, Sokoto, Nigeria. Niger J Clin Pract. 2017; 20(12): 1576–83. PubMed Abstract\n\nTilburt JC, Kaptchuk TJ: Herbal medicine research and global health: An ethical analysis. Bull World Health Organ. 2008; 86(8): 594–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarr KA, White TC, van Burik JA, et al.: Development of fluconazole resistance in Candida albicans causing disseminated infection in a patient undergoing marrow transplantation. Clin Infect Dis. 1997; 25(4): 908–10. PubMed Abstract \| Publisher Full Text\n\nTong SY, Davis JS, Eichenberger E, et al.: Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev. 2015; 28(3): 603–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrasad R, Balzi E, Banerjee A, et al.: All about CDR transporters: Past, present, and future. Yeast. 2019; 36(4): 223–233. PubMed Abstract \| Publisher Full Text\n\nKariuki S, Okoro C, Kiiru J, et al.: Ceftriaxone-resistant Salmonella enterica serotype typhimurium sequence type 313 from Kenyan patients is associated with the blaCTX-M-15 gene on a novel IncHI2 plasmid. Antimicrob Agents Chemother. 2015; 59(6): 3133–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMusila W, Kisangau D, Muema J: Conservation Status and Use of Medicinal Plants by Traditional Medical Practitioners in Machakos District, Kenya. 2001; (Odera 1997). Reference Source\n\nAnimal use in toxicity studies 9. Reference Source\n\nBeentje H, Adamson J, Bhanderi D: Kenya trees, shrubs, and lianas. National Museums of Kenya. 1994. Reference Source\n\nRevathy SS, Rathinamala R, Murugesan M: Authentication Methods for Drugs Used in Ayurveda, Siddha and Unani Systems of Medicine: An Overview. Int J Pharm Sci Res. 2012; 3(08): 2352–61. Reference Source\n\nKokate CK, Purohit AP, Gokhale SB: Pharmacognosy. Nirali Prakashan, Pune. 2006; 35. : 133–525.\n\nOECD Guideline For Testing Of Chemicals: Acute Oral Toxicity – Acute Toxic Class Method. 2001; 1–14. Reference Source\n\nOkumu M, Muia B, Mbaria JM, et al.: Photomicrographs of kidney and liver sections of mice treated with distilled water, extra virgin oil, and graded doses of aqueous and hexane root extracts of Fagaropsis hildebrandtii. figshare. Figure. 2019. http://www.doi.org/10.6084/m9.figshare.9199406.v2\n\nGuideline D, Of P, Test THE, Considerations I: OECD GUIDELINE FOR THE TESTING OF CHEMICALS. 2006; 2006: 1–11. Reference Source\n\n7. Randomisation. [cited 2019 Jul 16]. Reference Source\n\nYiaile AL, Mbaria JM, Ole-Mapenay IM, et al.: Preliminary Screening of Crude Extracts of Fagaropsis Angolensis for Anticancer Activity. Pharmacognosy Communications. 2018; 8(2): 75–80. Publisher Full Text\n\nManthey JA, Grohmann K, Guthrie N: Biological properties of citrus flavonoids pertaining to cancer and inflammation. Curr Med Chem. 2001; 8(2): 135–53. PubMed Abstract \| Publisher Full Text\n\nLiu H, Mou Y, Zhao J, et al.: Flavonoids from Halostachys caspica and their antimicrobial and antioxidant activities. Molecules. 2010; 15(11): 7933–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCushnie TP, Lamb AJ: Antimicrobial activity of flavonoids. Int J Antimicrob Agents. 2005; 26(5): 343–56. PubMed Abstract \| Publisher Full Text\n\nCowan MM: Plant products as antimicrobial agents. Clin Microbiol Rev. 1999; 12(4): 564–582. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGatsing D, Mbah JA, Garba IH, et al.: An antisalmonellal agent from the leaves of Glossocalyx brevipes Benth (Monimiaceae). Pak J Biol Sci. 2006; 9(1): 84–7. Publisher Full Text\n\nda Silva AP, Nascimento da Silva LC, Martins da Fonseca CS, et al.: Antimicrobial Activity and Phytochemical Analysis of Organic Extracts from Cleome spinosa Jaqc. Front Microbiol. 2016; 7: 963. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Oliveira YL, Nascimento da Silva LC, da Silva AG, et al.: Antimicrobial activity and phytochemical screening of Buchenavia tetraphylla (Aubl.) R. A. Howard (Combretaceae: Combretoideae). ScientificWorldJournal. 2012; 2012: 849302. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZdravkovic N, Baltic MZ, Starcevic M, et al.: Antimicrobial Activity of Thyme (Tymus vulgaris) and Oregano (Origanum vulgare) Essential Oils against Some Food-borne Microorganisms. Procedia Food Sci. 2015; 5: 18–21. Publisher Full Text\n\nNirogi R, Goyal VK, Jana S, et al.: What Suits Best for Organ Weight Analysis: Review of Relationship Between Organ Weight and Body / Brain Weight for Rodent Toxicity Studies. Int J Pharm Sci Res. 2014; 5(4): 1525–32. Publisher Full Text\n\nPorwal M, Khan NA, Maheshwari KK: Evaluation of Acute and Subacute Oral Toxicity Induced by Ethanolic Extract of Marsdenia tenacissima Leaves in Experimental Rats. Sci Pharm. 2017; 85(3): pii: E29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrasanth KM, Suba V, Ramireddy B, et al.: Acute and subchronic oral toxicity assessment of the ethanolic extract of the root of Oncoba spinosa (flacourtiaceae) in rodents. Trop J Pharm Res. 2015; 14(10): 1849–55. Publisher Full Text\n\nLee MY, Shin IS, Seo CS, et al.: Subchronic oral toxicity studies of the traditional herbal formula Bangpungtongseong-san in Crl: CD (SD) rats. J Ethnopharmacol. 2012; 144(3): 720–5. PubMed Abstract \| Publisher Full Text\n\nTeo S, Stirling D, Thomas S, et al.: A 90-day oral gavage toxicity study of D-methylphenidate and D,L-methylphenidate in Sprague-Dawley rats. Toxicology. 2002; 179(3): 183–96. PubMed Abstract \| Publisher Full Text\n\nAraújo MC, Barcellos NM, Vieira PM, et al.: Acute and sub chronic toxicity study of aqueous extract from the leaves and branches of Campomanesia velutina (Cambess) O. Berg. J Ethnopharmacol. 2017; 201: 17–25. PubMed Abstract \| Publisher Full Text\n\nOkumu MO, Ochola FO, Mbaria JM, et al.: Mitigative effects of Moringa oleifera against liver injury induced by artesunate-amodiaquine antimalarial combination in wistar rats. Clinical Phytoscience. 2017; 3(1): 18. Publisher Full Text\n\nDekant W, Vamvakas S: Biotransformation and membrane transport in nephrotoxicity. Crit Rev Toxicol. 1996; 26(3): 309–34. PubMed Abstract \| Publisher Full Text\n\nBurtis CA, Bruns DE: Tietz fundamentals of clinical chemistry and molecular diagnostics-e-book. Elsevier Health Sciences. 2014. Reference Source\n\nRavel R: Laboratório clínico: aplicações clínicas dos dados laboratoriais. In: Laboratório Clinico: aplicações clinicas dos dados laboratoriais. 1997. Reference Source\n\nGuidelines O, The FOR, Of T: Oecd guidelines for the testing of chemicals. 2013; 1–22. Reference Source\n\nOkumu M, Muia B, Mbaria JM, et al.: Underlying data (3) on the study titled 'Acute and sub-acute toxicity study of the root extracts of Fagaropsis hildebrandtii in mice and evaluation of their antimicrobial effects'. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9104894.v1\n\nOkumu M, Muia B, Mbaria JM, et al.: Underlying data (1) on the study titled 'Acute and sub-acute toxicity study of the root extracts of Fagaropsis hildebrandtii in mice and evaluation of their antimicrobial effects'. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9101231.v2\n\nOkumu M, Muia B, Mbaria JM, et al.: Underlying data (2) on the study 'Acute and sub-acute toxicity study of the root extracts of Fagaropsis hildebrandtii in mice and evaluation of their antimicrobial effects'. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9104480.v3\n\nOkumu M, Muia B, Mbaria JM, et al.: Extended data on the study titled 'Acute and sub-acute toxicity study of the root extracts of Fagaropsis hildebrandtii in mice and evaluation of their antimicrobial effects'. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9083348.v1" }	[ { "id": "54119", "date": "07 Oct 2019", "name": "Mekbeb Afework", "expertise": [ "Reviewer Expertise Toxicological effects of herbal medicines on hematological and biochemical parameters and histopathology of vital organs including liver and kidneys." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very good and up to the standard study that comprehensively investigated the acute and subacute toxicity and antimicrobial effects of the root extract of Fagaropsis hildebrandtii which is a widely used herbal medicine. It has a significant contribution towards the currently increasing search for safe, affordable and effective drugs of plant origin and we approve it. However, we have the following comments which would have been accommodated/explained. These include:\nHow was the starting doses of the extract (as 300 mg/kg for acute and 250 mg/kg for sub-acute) determined. Why was LD50 not determined?\n\nWhen and during what season was the herb collected and the study conducted?\n\nThe root of the plant is mentioned used traditionally as boiled. However, the investigation was carried out on fresh root extract. Why not the boiled ones same as traditionally used? or do the authors believe heat has no effect on the active ingredients of the plant?\n\nWhat could be the cause for the death of the female mice treated with the extract at the highest does (1000mg/kg body wt)? Why macroscopic and microscopic pathological observations were not done? At what date(s) did the mice die?\n\nThe description of histopathological observations made on page 10 appears an abridged version of figure legends. There are also statements that do not go along with the figure legends as pointed out below:\n\nIn Figure 5 micrographs for histopathology of kidney, only 4 pictures are presented. However, in the text, page 10, seven pictures (A-G) are described, and does not go along.\n\nIn Figure 6 micrographs for histopathology of liver.\n\nPicture “A” appears as at a lower magnification as compared to “B-F”, but is indicated as x400 same as others, and what B is meant to indicate is not stated.\n\nPicture “C” in the figure legend is stated to show diffuse cloudy swelling\n\n(CS) of the hepatocytes, but mentioned as normal same as A & B in the text, page 10.\nWe suggest these should be corrected in the text and better be briefly stated in a manner of reporting the observations.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5019", "date": "07 Feb 2020", "name": "Beatrice Muia", "role": "Author Response", "response": "We are grateful to the reviewers for taking the time to provide feedback on our manuscript. We wish to respond to each of their queries as below; 1. Selection of the starting doses and determination of LD50 The choice of 300 mg/kg as a starting dose in evaluating the acute toxicity of the root bark extracts of F. hildebrandtii was informed by the Organization for Economic Cooperation and Development (OECD) test guidelines no 423 (1). According to these guidelines, in cases where there is no information on the toxicity of a substance, it is recommended to use a starting dose of 300 mg/kg body weight for animal welfare reasons (1). The choice of 250 mg/kg as a starting dose in evaluating the sub-acute toxicity of root bark extracts of F. hildebrandtii was informed by OECD test guidelines no 407 (2). According to these guidelines, at least three test groups, and a control group should be used and a limit dose of up to 1000mg/kg is considered (2). Thereafter, a descending sequence of dose levels should be selected to demonstrate any dose-related responses and no-observed-adverse effects at the lowest dose (NOAEL) (2). In view of this, the sequential dose level we selected was 1000mg/kg, 500mg/kg, and 250mg/kg. Unfortunately, unlike other methods of LD50 determination, it is only possible to determine a precise LD50 using OECD guidelines when at least two of the tested doses result in mortality greater than 0% and lower than 100% (1). In our case, none of the doses tested resulted in the death of any mice. In such a case, the OECD guidelines allow for the determination of defined exposure ranges where lethality may be expected (1). This is what we have provided in reporting the acute oral toxicity of the extracts (i.e. LD50 >2000mg/kg). 2. The season of plant collection and when the study was conducted. Roots of F. hildebrandtii were collected during the hot dry season in February 2017. Acute and sub-acute studies were conducted between January 2018 and February 2018. Before this period, extraction, phytochemical analysis, and evaluation of the effect of the extracts on some human pathogens had been evaluated (April 2017 to December 2017). 3. Why the investigations were carried out on fresh root extracts rather than boiled root extracts. This was done to preserve the integrity of the phytochemicals conferring antimicrobial activity to our plant of interest. According to a 2013 study on the antibacterial activity of selected medicinal plants against some common human pathogens, cold water extracts exhibit better inhibitory effects against S. aureus, B. subtilis, P. vulgaris, P. aeruginosa, and E.coli than hot water extracts (3). Based on the OECD test guidelines, female rodents (rats and mice) are usually more susceptible to toxic compounds than their male counterparts (1). Therefore, it could be argued that the physiological (biochemical, hematological) and pathological alterations we observed were more severe in female mice than their male counterparts. The metabolic roles of the kidney and liver may have exposed these organs to dose-dependent toxic effects. The mice died on weeks 3 and 4 (Between February 2th 2018, and February 17th, 2018) 4. The discrepancy between the figure legends of liver and kidney sections and the description provided in the text.a. Thank you for your observation. We have rectified the in-text description to better reflect the figure legends provided in Figures 5 and 6. We wish to sincerely thank the reviewers for taking the time to provide feedback on this manuscript. REFERENCES1. Guideline O, Testing FOR, Chemicals OF. Acute Oral Toxicity – Acute Toxic Class Method. 2001;(December):1–14.2. Guidelines O. Repeated dose 28-day oral toxicity study in rodents. Oecd Guidelines for the Testing of Chemicals. 2008;(October):1–13.3. Khan UA, Rahman H, Niaz Z, Qasim M, Khan J, Tayyaba, et al. Antibacterial activity of some medicinal plants against selected human pathogenic bacteria. European Journal of Microbiology and Immunology. 2013;3(4):272–4." } ] } ]	1	https://f1000research.com/articles/8-1444
https://f1000research.com/articles/8-237/v1	28 Feb 19	{ "type": "Research Note", "title": "National culture as a correlate of research impact and productivity", "authors": [ "Juneman Abraham" ], "abstract": "National culture has been overlooked in discussions related to research productivity and impact owing to individual, socio-political structure, and economic factors. This study shows the relationships between the dimensions of cultural value orientation of the nation and research performance indicators. More than 60 countries were included and Pearson correlation analysis was employed. The variables were taken from Geert Hofstede and Scimago Journal & Country Rank worksheets. This study found that (1) Individualism has significant correlations with the majority of the indicators; (2) Power distance and indulgence correlate with a country’s research impact in the form of citation per document; (3) Masculinity, long term orientation, and uncertainty avoidance do not correlate with the indicators. Owing to the fact that the national culture is relatively enduring, countries need to measure their elasticity of hopes and action plans in an effort to boost research productivity and impact, by integrating the national culture in the estimate.", "keywords": [ "research impact", "research productivity", "national culture", "individualism", "indulgence", "power distance", "citations per document", "self citations" ], "content": "Introduction\n\nMakri (2018) recently released a report on the increasing number of publications in various countries. She stated that it’s unclear what has triggered and driven the strong gains in Egypt and Pakistan. Throughout the report, various variables believed to be responsible for the increasing number of publications, such as indexation duration, funding, global engagement, international collaboration, and political policies on science and higher education, are explained.\n\nSeveral predictors of research productivity and impact had been identified, i.e. author characteristics, co-authorship networks, citation history, journal impact factors, twits (Xiaomei et al., 2017), cohort effects (in terms of scientific discipline), age, career stages, gender, the country of origin of the PhD holders, and reward structure of the research enactment (Claudia & Francisco, 2007). They are mostly at the individual and institutional level. At the country level, the predictors are the number of universities, GDP per capita, control of corruption, civil liberties (Mueller et al., 2016), country’s wealth and population size, country’s value of research tradition, tenure and promotion criterion, experimental costs, IRB (Institutional Review Boards) review flexibility, language barrier, and the training of new young researchers (Demaria, 2009).\n\nHowever, national cultural orientation is yet to be analyzed, with the present study assuming that individual, institutional, and structural factors are also influenced by the cultural values of a nation. Hofstede Insights (2019) defined culture as the collective mental programming of the human mind which distinguishes one group of people from another, consisting of six dimensions, i.e. (1) power distance (PD) – acceptance on the unequal power distribution in a society; (2) uncertainty avoidance (UA) – intolerance of ambiguity and uncustomary thoughts and practices; (3) individualism (IND) – projection of individuals’ “I” in a society rather than “we” (collectivism); (4) masculinity (MAS) – the toughness and competitiveness rather than the tenderness and cooperativeness (femininity) orientation; (5) long term orientation (LTO) – the society’s preference of time-honored rather than pragmatic approaches (short term normative orientation); and (6) indulgence (IVR) – the society facilitation towards a fun and enjoyable life rather than restraint (suppression of needs gratification by strict social norms).\n\nNational culture is relatively stable (Maseland & van Hoorn, 2017) and is widely used to explain various performances at the country level, such as learning and academic performance (Signorini et al., 2009). The present study hypothesized that there are correlations between the national culture dimensions and research performance indicators. The research performance is assumed to be mediated by research culture, and the culture experiences stimulations and challenges from the national culture; as happened in China, the bureaucratic (high power distance) and nepotistic culture suppresses an innovative and meritocratic research culture (Shi & Rao, 2010).\n\n\nMethods\n\nAll following data were retrieved on December 18, 2018 and compiled into a worksheet (see Underlying data (Abraham, 2019)) as the material of this present analysis. Countries’ research impact (citations per document/CPD, citations, self-citations) and productivity (total documents) were obtained from the Scimago Journal & Country Rank (https://www.scimagojr.com/countryrank.php?out=xls), while national cultural orientations (pdi=power distance, idv=individualism, mas=masculinity, uai=uncertainty avoidance, ltowvs=long term orientation, ivr=indulgence) were acquired from Geert Hofstede web site (https://geerthofstede.com/wp-content/uploads/2016/08/6-dimensions-for-website-2015-08-16.xls). Pearson product-moment correlation analysis was conducted using IBM SPSS Statistics version 20 for Windows.\n\n\nResults\n\nThe descriptive and correlational statistics are presented in Table 1 and Table 2. As shown, among 68 countries, IND is positively correlated with CPD (r=0.506, p<0.01), total documents (r=0.351, p<0.01), citations (r=0.405, p<0.01), and self-citations (r=0.304, p<0.05). MAS, UA, and LTO do not correlate with these four. PD (r=-0.555, p<0.01, N=68) and IVR (r=0.480, p<0.01, N=91) correlate with CPD. Total documents, citations, and self-citations correlate with each other with r>0.90, p<0.01, N=239. CPD is positively, but weakly, correlated with total documents, total citations and total self-citations, with r<0.20, p<0.05, N=239.\n\nDOC = Total documents; CIT = Total citations; SELF = Total self-citations; CPD = Citations per document; PD = Power distance; IND = Individualism; MAS = Masculinity; UA = Uncertainty avoidance; LTO = Long term orientation; IVR = Indulgence\n\nDOC = Total documents; CIT = Total citations; SELF = Total self-citations; CPD = Citations per document; PD = Power distance; IND = Individualism; MAS = Masculinity; UA = Uncertainty avoidance; LTO = Long term orientation; IVR = Indulgence\n\n*. Correlation is significant at the 0.01 level (2-tailed).\n\n. Correlation is significant at the 0.05 level (2-tailed).\n\nAmong 54 countries, even after controlling the Log GDP per capita (taken from the World Happiness Report), the correlations between IND and CPD (r=0.439, p<0.01), total documents (r=0.268, p<0.05), and citations (r=0.320, p<0.05), between PD and CPD (r=-0.504, p<0.01), as well as between IVR and CPD (r=0.411, p<0.01) persist (see Table 3).\n\nDOC = Total documents; CIT = Total citations; SELF = Total self-citations; CPD = Citations per document; PD = Power distance; IND = Individualism; MAS = Masculinity; UA = Uncertainty avoidance; LTO = Long term orientation; IVR = Indulgence\n\n\nDiscussion\n\nThe positive correlations between IND and CPD, total documents, citations, and self-citations could be explained using the findings of Deschacht & Maes (2017). They found that in countries with more individualistic cultures: (1) the scientists prioritize their self-development, (2) the records of scientific work are historically longer (usually Western countries), and (2) self-citations flourish more. This does not necessarily mean that there have been citation abuses, but that self-citation is used to refer to their prior works, thereby, preventing unnecessary repetitions of ideas in newer works (Deschacht, 2017). Although IND and collaboration are often contested (e.g. Kemp, 2013), a “collaborative individualism” (Limerick & Cunnington, 1993) – stressing both working together and self-emancipation – is possible, explaining the positive correlation.\n\nPD has a negative correlation with CPD. In this case, PD might manifest itself in academic writing in the form of rigid, authoritative, defensive, and dogmatic styles (Koutsantoni, 2005). In addition, PD negatively correlates with democracy (Maleki & Hendriks, 2014). The lower level of democracy reduces the opportunity of the academic community to exchange and market (in the broad sense) scientific information, as well as debate openly. Likewise, democracy that does not flourish deters the use of research results in creating public policies. In addition, science is co-opted or used as just a tool to achieve exclusive interests by ideologues, pundits and political leaders; they ignore the state-of-the-art nature of the research (Branscomb & Rosenberg, 2012). All the conditions could reduce CPD.\n\nIVR is positively correlated with CPD; this may be because IVR facilitates academic freedom (Ohmann, 2011) and manifests itself in a “lovely” academic writing style (Kiriakos & Tienari, 2018). This style is not dry and cold, but rather dialogical, humanistic, more reflexive, and capable of showing authors’ courage and vulnerability. Compelling insights are more easily born from the writings that embody those qualities; as mentioned, “a thin line exists between interesting insights and self-indulgence” (Nadin & Cassell, 2006, p. 214). Scientific authors who read such works would be attracted to cite them, leading to an increase in the works’ CPD. In addition, “strategic indulgence” is possible and known to be a creative process that enables one to balance academic activity (such as writing) with non-academic ones (Jia et al., 2018) – fostering insight.\n\nThe weak correlation between CPD and other research performance indicators shows that CPD is more difficult to manipulate or be an object of the author’s “engineering”.\n\n\nConclusion\n\nNational culture dimensions, especially individualism, power distance, and indulgence, are pivotal variables that are to be considered in justifying research impact and productivity. National culture can be integrated as a moderating variable in the predictive relationship between GDP per capita and research impact and productivity. Diversification of this study – based on the document and authors’ collaboration types, the indexing databases, the disciplines, the open science practices, as well as the history and development of the research in a country – is a future opportunity for further study.\n\n\nData availability\n\nGeert Hofstede: Dimension data matrix. https://geerthofstede.com/research-and-vsm/dimension-data-matrix/ (Hofstede et al., 2010)\n\nScimago Journal & Country Rank: Download data. https://www.scimagojr.com/countryrank.php?out=xls (Scimago Lab, 2018)\n\nWorld Happiness Report 2018: Chapter 2: online data. https://s3.amazonaws.com/happiness-report/2018/WHR2018Chapter2OnlineData.xls (Helliwell et al., 2018)\n\nAll source data was accessed and retrieved on the 18/12/2018\n\nFigshare: National culture, research performance indicators, and log GDP Per capita. https://doi.org/10.6084/m9.figshare.7723211 (Abraham, 2019)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThe authors declared that no grants were involved in supporting this work\n\n\nReferences\n\nAbraham J: National culture, research performance indicators, and log GDP per capita. [Data set]. Figshare. 2019. http://www.doi.org/10.6084/m9.figshare.7723211\n\nBranscomb LM, Rosenberg AA: Science and democracy. 2012. Reference Source\n\nClaudia GB, Francisco MV: The determinants of research output and impact: A study of Mexican researchers. Res Policy. 2007; 36(7): 1035–1051. Publisher Full Text\n\nDemaria A: Research productivity among nations. J Am Coll Cardiol. 2009; 54(25): 2460–2462. PubMed Abstract \| Publisher Full Text\n\nDeschacht N, Maes B: Cross-cultural differences in self-promotion: A study of self-citations in management journals. J Occup Organ Psychol. 2017; 90(1): 77–94. Publisher Full Text\n\nDeschacht N: Me, myself, and I: self-citation rates are higher in individualist cultures than in collectivist cultures. 2017. Reference Source\n\nHelliwell J, Layard R, Sachs J: World Happiness Report 2018. Sustainable Development Solutions Network, New York, 2018. Reference Source\n\nHofstede G, Hofstede GJ, Minkov M: Cultures and Organizations: Software of The Mind. McGraw Hill, New York, 2010. Reference Source\n\nHofstede Insights: National culture. 2019. Reference Source\n\nJia L, Hirt ER, Koh AH: How to have your cake and eat it too: strategic indulgence in big-time collegiate sports among academically successful students. Soc Psychol Personal Sci. 2018. Publisher Full Text\n\nKemp AT: Collaboration vs. individualism: what is better for the rising academic? Qual Rep. 2013; 18(50): 1–8. Reference Source\n\nKiriakos CM, Tienari J: Academic writing as love. Manag Learn. 2018; 49(3): 263–277. Publisher Full Text\n\nKoutsantoni D: Greek cultural characteristics and academic writing. J Mod Greek Stud. 2005; 23(1): 97–138. Reference Source\n\nLimerick D, Cunnington B: Collaborative Individualism and The End of The Corporate Citizen. In: Limerick D, Cunnington B (eds). Managing The New Organisation. Chapter 4. Business and Professional Publishing, Chatswood, 1993. Reference Source\n\nMakri A: Pakistan and Egypt had highest rises in research output in 2018. Nature. 2018. Publisher Full Text\n\nMaleki A, Hendriks F: The relation between cultural values and models of democracy: a cross-national study. Democratization. 2014; 22(6): 981–1010. Publisher Full Text\n\nMaseland R, van Hoorn A: Culture at the country level. In: van Herk H, Torelli CJ (eds.). Cross Cultural Issues in Consumer Science and Consumer Psychology. Springer, New York, 2017; 7–32. Publisher Full Text\n\nMueller CE, Gaus H, Konradt I: Predicting research productivity in international evaluation journals across countries. J Multidiscip Eval. 2016; 12(27): 79–92. Reference Source\n\nNadin S, Cassell C: The use of a research diary as a tool for reflexive practice: Some reflections from management research. Qualitative Research in Accounting & Management. 2006; 3(3): 208–217. Publisher Full Text\n\nOhmann R: Academic freedom’s best days. Inside Higher Ed. 2011. Reference Source\n\nScimago Lab: Scimago journal & country rank. [Data set]. 2018. https://www.scimagojr.com/countryrank.php\n\nShi Y, Rao Y: China’s research culture. Science. 2010; 329(5996): 1128. PubMed Abstract \| Publisher Full Text\n\nSignorini P, Wiesemes R, Murphy R: Developing alternative frameworks for exploring intercultural learning: a critique of Hofstede's cultural difference model. Teach High Educ. 2009; 14(3): 253–264. Publisher Full Text\n\nXiaomei B, Hui L, Fuli Z, et al.: An overview on evaluating and predicting scholarly article impact. Information. 2017; 8(3): 73. Publisher Full Text" }	[ { "id": "46349", "date": "28 Mar 2019", "name": "Jonathan P. Tennant", "expertise": [ "Reviewer Expertise Palaeontology", "Open Scholarly Communication" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author presents an interesting piece of insight into research impact through a cultural lens, which is quite distinct from a lot of more recent studies which tend to focus on ‘academic impact’ metrics. As a short note, I found it useful in exposing a different dimension to the ongoing debates around research impact. Given my area of “expertise”, I feel that someone who understands social research impact more could provide a great deal of additional insight here during the review process.\nData\nThe data are included in Figshare, as well as summarized in integrated tables. I note that there are a lot of missing data included though, is this just a case of availability? Also, I note that the Scimago database is based on Scopus data, which tends to be biased in a number of dimensions. Is it possible to make a note of this?\nAbstract\nThe abstract jumps right into results around Individualism and Power distance and indulgence, without describing what these are (even briefly). This makes it difficult to understand for readers who are perhaps unfamiliar with these concepts. Perhaps a brief explanation of these could be added instead of describing the methods and the data sources, which aren’t really needed?\nIntroduction\nJust to pull out the ‘correlation does not imply causation’ card here; just because there is a correlation between number of publications and other external factors, does not imply a causal relationship necessarily. There are a couple of typos (e.g.‘twits’) that might just need a quick copy edit to fix. I think the Introduction does a nice job of describing the previous research, and situates the present report well within that. Not sure if the comment about China at the end of the Introduction adds too much here.\nMaterials and methods\nSo the methods are pretty simple, which is nice. But also, I think perhaps a bit too simple here given that you’re performing a lot of bivariate analyses, and a couple of extra steps are recommended.\nFirst, you want to perform an assessment of normality for data series prior to any correlation analyses, using the Shapiro–Wilk test (e.g.,shapiro.test function in R). From the output, if the p-values are greater than the pre-defined alpha level (traditionally, 0.05) this implies that the distribution of the data are not significantly different from a normal distribution, and therefore you can assume normality and use Pearson’s test (Pearson’s product moment correlation coefficient [r]). If p > 0.05, you should instead perform a non-parametric Spearman’s rank correlation (ρ). Secondly, once you’ve done this, for each test, report both the raw and adjusted p-values. The latter can be calculated using the p.adjust() function, and using the ‘BH’ model (Benjamini & Hochberg, 19951). This method accounts for the false-discovery test when performing multiple hypothesis tests with the same data set, which can inflate type-1 error (i.e. in order to avoid falsely rejecting a true null hypothesis; a false positive). What this will probably do is reduce the ‘significance’ of some of your results too (which is why it’s best to report both the raw and adjusted values).\n\nIn addition to this, it seems like you have multivariate data, so multivariate analyses might be more informative here. I would strongly recommend performing a Principal Components Analysis on your data (perhaps just only with the variables with more complete data), and inspecting that as a compliment to the bivariate ones. This is fairly easy to do and display using in built functions in R.\nResults\nI expect that the results will change a bit given my above recommendations to the methods, so won’t comment too much on them at this stage. The nice thing about PCA though is that it produces good summary plots, which might be useful here. In the text, can the country abbreviations be given to make reading a bit easier? M, SD, and N I think need explaining here too. Lots of acronyms can get a bit confusing!\nDiscussion and conclusions\nAs above, I don’t want to comment too much on the Discussion and Conclusions at the present, as I think the above recommended methods will change some of the interpretations. However, at the present there seems to be a logical progression between reported results and conclusions.\nCongratulations to the author on a great and interesting piece of work. I would be happy to see a revised version of this too if needed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "46347", "date": "15 Apr 2019", "name": "Ludo Waltman", "expertise": [ "Reviewer Expertise I am an expert in scientometrics. I don't have any specific expertise on the cultural orientation of countries." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a short paper that presents a brief but easy-to-understand analysis of the correlation between the cultural orientation of countries and countries’ scientific performance in terms of bibliometric statistics.\n\nIt would be helpful if the author could offer more extensive information on the way in which the variables used in the analysis have been obtained. The definitions of the bibliometric variables need to be carefully explained. Likewise, it should be explained how the variables for national cultural orientations have been obtained.\n\nThe statistical analysis is carried out using Pearson correlation analysis. Before presenting the correlations, the author should present descriptive statistics for the variables included in the analysis. In particular, I would like to know whether some of the variables may be highly skewed. If this is the case, the use of Pearson correlation analysis could easily lead to misleading results. The use of for instance the Spearman correlation may then be more appropriate.\n\nThe author relies strongly on statistical significance testing. My recommendation is to leave out all significance tests and instead to present confidence intervals for the correlation coefficients. Significance testing leads to problematic dichotomous thinking, as has for instance been pointed out in a recent contribution in Nature (Amrhein et al.1). Following the so-called estimation statistics approach, reporting confidence intervals is preferable over reporting significance tests (https://en.wikipedia.org/wiki/Estimation_statistics). I am aware that another reviewer (Tennant) recommends performing even more significance tests. I disagree with this recommendation. I don’t consider this to be good statistical practice.\n\nIt would be nice if the author could deepen the analysis a bit more. This can for instance be done by showing scatter plots for the most interesting relationships between variables. In these scatter plots, the names of countries could be shown, especially for those countries that seem to display interesting behavior (e.g., outliers). This would lead to a more in-depth analysis that probably offers richer insights.\n\nThe paper uses lots of abbreviations. This makes the paper more difficult to read. My recommendation is to reduce the number of abbreviations that are used. It may also be helpful to include a table listing all abbreviations and the corresponding full terms.\n\nThe author interprets the analysis in terms of correlation instead of causation. This is very good. There is a risk, however, that some readers may give causal interpretations to the findings of the author. My suggestion is to add a sentence at the end of the paper emphasizing that causal interpretations are not warranted.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-237
https://f1000research.com/articles/8-1760/v1	16 Oct 19	{ "type": "Study Protocol", "title": "Investigation of Risk Of Bias due to Unreported and SelecTively included results in meta-analyses of nutrition research: the ROBUST study protocol", "authors": [ "Matthew J. Page", "Lisa Bero", "Cynthia M. Kroeger", "Zhaoli Dai", "Sally McDonald", "Andrew B. Forbes", "Joanne E. McKenzie", "Lisa Bero", "Cynthia M. Kroeger", "Zhaoli Dai", "Sally McDonald", "Andrew B. Forbes", "Joanne E. McKenzie" ], "abstract": "Background: Dietary guidelines should be informed by systematic reviews (SRs) of the available scientific evidence. However, if the SRs that underpin dietary guidelines are flawed in their design, conduct or reporting, the recommendations contained therein may be misleading or harmful. To date there has been little empirical investigation of bias due to selective inclusion of results, and bias due to missing results, in SRs of food/diet-outcome relationships. Objectives: To explore in SRs with meta-analyses of the association between food/diet and health-related outcomes: (i) whether systematic reviewers selectively included study effect estimates in meta-analyses when multiple effect estimates were available; (ii) what impact selective inclusion of study effect estimates may have on meta-analytic effects, and; (iii) the risk of bias due to missing results (publication bias and selective non-reporting bias) in meta-analyses. Methods: We will systematically search for SRs with meta-analysis of the association between food/diet and health-related outcomes in a generally healthy population, published between January 2018 and June 2019. We will randomly sort titles and abstracts and screen them until we identify 50 eligible SRs. The first reported meta-analysis of a binary or continuous outcome in each SR (the ‘index meta-analysis’) will be evaluated. We will extract from study reports all study effect estimates that were eligible for inclusion in the index meta-analyses (e.g. from multiple instruments and time points) and will quantify and test for evidence of selective inclusion of results. We will also assess the risk of bias due to missing results in the index meta-analyses using a new tool (ROB-ME). Ethics and dissemination: Ethics approval is not required because information will only be extracted from published studies. Dissemination of the results will be through peer-reviewed publications and presentations at conferences. We will make all data collected from this study publicly available via the Open Science Framework.", "keywords": [ "Bias", "Publication bias", "Systematic reviews", "Diet", "Food and Nutrition" ], "content": "Introduction\n\nSuboptimal diet is one of the leading contributors to mortality and morbidity globally1. Dietary guidelines, which provide recommendations on types and amounts of foods to consume and dietary patterns to adopt, are developed with the aim of reducing non-communicable disease attributable to diet. Systematic reviews (SRs) of the available scientific evidence often underpin dietary guidelines2. However, if the SRs are flawed in their design, conduct or reporting, the recommendations contained therein may be misleading or harmful.\n\nBias in the results of SRs can arise through various processes3. Systematic reviewers often face a multiplicity of results for particular outcomes in the included studies (e.g. there may be results for weight loss at multiple time points, each of which are presented unadjusted and adjusted for prognostic factors, such as age and sex)4,5. When multiple results for an outcome are available in study reports, systematic reviewers’ choice about which result to include in a meta-analysis may be influenced by the P value, magnitude or direction of the result – this is known as ‘selective inclusion of results’6. For example, instead of including the result for weight loss that arose from the time point considered a priori to be the most clinically important, or which was adjusted for the most appropriate set of prognostic factors, systematic reviewers may select a result simply because it was the largest in magnitude, or had the smallest P value.\n\nBias in meta-analyses can arise not only when systematic reviewers selectively include results, but also when results of some eligible studies are unavailable for inclusion7. There is extensive evidence that shows many studies are never published8, and that those studies that are published tend to have larger effect estimates than unpublished studies9. The term ‘publication bias’ has often been used to describe this problem. In addition, published studies often omit results for some of the outcomes that were measured10, with reported results more likely to be statistically significant than non-reported results11. The terms ‘selective outcome reporting’ and ‘outcome reporting bias’ have been used to describe this problem, but we prefer the term ‘selective non-reporting bias’ as it emphasises the non-reporting of study results. Regardless of whether an entire study report or a particular study result is unavailable selectively (e.g. because the P value, magnitude or direction of the results were considered less favourable by the investigators), the consequence is bias in a meta-analysis because available results differ systematically from missing results. The term ‘bias due to missing results’ has recently been coined to describe the bias in meta-analyses that arises from non-publication or non-reporting of study results12.\n\nThere has been little empirical investigation of bias due to selective inclusion of results and bias due to missing results in SRs with meta-analyses of the association between food/diet and health-related outcomes. The only known investigation of selective inclusion of results focused on meta-analyses of randomized trials of interventions for arthritis or depressive or anxiety disorders13. Also, previous assessments of reporting biases in nutrition research have been limited to an exploration of publication bias in meta-analyses of the association between diet and cardiovascular disease or mortality14. There has been no formal investigation of selective non-reporting of results in studies included in meta-analyses of food/diet-outcome relationships.\n\nTherefore, the aim of this research is to investigate various biases in SRs with meta-analyses of the association between food/diet and health-related outcomes. The objectives are to explore:\n\n(i) whether systematic reviewers selectively included study effect estimates in meta-analyses when multiple effect estimates were available;\n\n(ii) what impact selective inclusion of study effect estimates may have on meta-analytic effects, and;\n\n(iii) the risk of bias due to missing results in meta-analyses.\n\n\nMethods\n\nWe will systematically search for SRs with meta-analysis of the association between food/diet and health-related outcomes in a generally healthy population, published between January 2018 and June 2019. We will randomly sort titles and abstracts and screen them until we identify 50 eligible SRs. The first reported meta-analysis of a binary or continuous outcome in each SR (which we refer to as the ‘index meta-analysis’) will be evaluated. We will extract from study reports all study effect estimates that were eligible for inclusion in the index meta-analyses (e.g. from multiple instruments and time points), and will calculate a statistic – the Potential Bias Index15 – to quantify and test for evidence of selective inclusion of results. The risk of bias due to missing results (arising from publication bias and selective non-reporting bias) in the index meta-analyses will also be assessed using a new tool (the Risk Of Bias due to Missing Evidence (ROB-ME) tool12).\n\nWe will seek a sample of SRs with meta-analysis meeting the following criteria:\n\nincludes studies of people of any ages (i.e. infants, children, adolescents, adults or elderly people) and backgrounds in the generally healthy population, including pregnant and breastfeeding women and people with common diet-related risk factors such as being overweight or having high blood pressure;\n\nincludes randomized trials or non-randomized studies evaluating the effects of at least one type of food (e.g. whole grains, fruit) or at least one food-defined dietary pattern (e.g. high intake of processed meat) on any binary (e.g. mortality) or continuous (e.g. weight) health-related outcome;\n\npublished from 1 January 2018 to 30 June 2019;\n\nwritten in English;\n\nincludes citations for all studies included in the SR; and\n\npresents the summary statistics or effect estimate and its precision (e.g. 95% confidence interval) for each included study, and the meta-analytic effect estimate and its precision, for at least one meta-analysis of a binary or continuous outcome.\n\nWe will adopt the definition of “systematic review” used in the 2019 edition of the Cochrane Handbook for Systematic Reviews of Interventions: “A systematic review attempts to synthesize all empirical evidence that fits pre-specified eligibility criteria in order to answer a specific research question. It uses explicit, systematic methods that are selected with a view to minimizing bias, thus providing more reliable findings from which conclusions can be drawn and decisions made”16. We will use the criteria adopted by Page et al. to identify articles as SRs with meta-analysis, i.e. we will include articles with explicitly stated methods of study identification (e.g. a search strategy), explicitly stated methods of study selection (e.g. eligibility criteria and selection process), and a meta-analysis of study results17. We will not exclude articles based on the level of detail about the methods provided (e.g. articles with a line-by-line Boolean search strategy or just a list of the key words used in the bibliographic databases will both be considered to meet the criteria for an SR).\n\nWe will exclude:\n\nSRs that did not include any meta-analysis of a binary or continuous outcome;\n\nmeta-analyses or pooled analyses of studies conducted outside the context of a SR (e.g. when individual participant data are combined from a set of cohort studies outside the context of a systematic review, e.g. see Zhong et al.18);\n\nSRs that focus only on nutrient-specific associations with outcomes (e.g. those examining the effects of single nutrients such as folic acid, salt).\n\nSRs including studies that were restricted to people with a health condition (e.g. type II diabetes), people who are obese, or frail elderly people who are at risk of malnutrition (by ‘frail’ we mean a person who, following a minor stress, experiences a large deterioration in function and does not return to baseline homeostasis19); and\n\nSRs that were co-authored by a member of the investigator team, because the assessment of bias may be influenced by the investigators’ prior involvement in the SR.\n\nWe will identify eligible SRs by searching PubMed and Epistemonikos (a database of SRs and other syntheses of health research that have been identified via systematic searches of the Cochrane Database of Systematic Reviews, PubMed, EMBASE, CINAHL, PsycINFO, LILACS, DARE, Campbell Library, JBI Database and EPPI-Centre Evidence Library). In PubMed, we will use the search strategy for diet- or nutrition-related studies developed and validated by Durao et al.20, combine this strategy with the PubMed filter for meta-analyses, and restrict the search from 1 January 2018 to 30 June 2019 (search strategy as extended data21). In Epistemonikos, we will use the following search strategy: (title:((nutri* OR diet)) OR abstract:((nutri OR diet*))) and limit the search from 1 January 2018 to 30 June 2019.\n\nSelection of SRs. We will export titles and abstracts into Microsoft Excel, remove all duplicate records, and randomly sort the remaining records. In the piloting phase, four investigators (MJP, CMK, ZD and SM) will independently assess 50 abstracts against the inclusion criteria (rating each as ‘Eligible’, ‘Ineligible’, or ‘Unsure’), to ensure the criteria are applied consistently. Following piloting, two investigators (MJP and one of CMK, ZD or SM) will independently screen titles and abstracts of 450 records. The full text of records rated as ‘Eligible’ or ‘Unsure’ will then be retrieved and assessed independently against the inclusion criteria by two investigators (MJP and one of CMK, ZD or SM). We will repeat this process (screening batches of 500 records) until the target sample of 50 SRs is met. Any discrepancies in screening decisions at each stage will be resolved via discussion, or by consultation with another investigator (JM or LB) where necessary.\n\nSelection of index meta-analyses. From each SR one investigator (MJP) will select one meta-analysis of a binary or continuous outcome for assessment, stratifying the selection so that the final sample includes 25 binary and 25 continuous outcome meta-analyses. The selected meta-analysis will be the first meta-analytic result that is presented in the SR, and henceforth is referred to as the ‘index meta-analysis’. The index meta-analysis may be selected from the abstract, summary of findings table, or results section of the SR, depending on where the meta-analytic result is first reported in the publication. We will include meta-analyses regardless of the outcome domain measured (e.g. all-cause mortality, diabetes incidence), meta-analytic effect measure (e.g. odds ratio, mean difference), meta-analytic model (e.g. fixed-effect, random-effects), approach to modelling (i.e. frequentist or Bayesian) and number and type of included studies (i.e. randomized trial or non-randomized study). We will select only standard pairwise meta-analyses of aggregate data, not dose-response meta-analyses, network meta-analyses or meta-analyses of individual participant data.\n\nWe will retrieve all reports of studies that were included in each index meta-analysis, as cited by the systematic reviewers. Study reports could include journal articles, conference abstracts, dissertations, trial results posted in trials registers, or any other reports (e.g. government reports). If more than one reference for a study was cited by the systematic reviewers (e.g. a study was reported in multiple journal articles, or in a journal article and a conference abstract), we will retrieve all references cited. If study reports are written in languages other than English, we will attempt to translate them using Google Translate; we will exclude reports if the translation is not interpretable. We will also retrieve reports of studies that were eligible for inclusion in the meta-analysis but excluded by the systematic reviewers, to explore whether any eligible outcome data may have been missed from these reports or potentially excluded because of the nature of the results (e.g. statistical non-significance).\n\nWe will collect data using a standardised form with detailed guidance created in REDCap (Research Electronic Data Capture), a secure, web-based software platform designed to support data capture for research studies22,23. Four investigators (MJP, CMK, ZD and SM) will initially pilot the data collection form on a random sample of five index meta-analyses and all of their included studies, to ensure consistency in the data collection. Any discrepancies will be discussed amongst all four investigators, and the form and guidance will be revised as necessary. Following piloting, two investigators (MJP and one of CMK, ZD or SM) will independently collect data from all remaining index meta-analyses and included studies. Any discrepancies between the data extracted will be resolved through discussion or adjudication by a third investigator (JM or LB) if necessary.\n\nData items to describe the general characteristics of the SRs. We will record the following characteristics of each of the included SRs:\n\nJournal it is published in;\n\nYear of publication;\n\nCountry of corresponding author of the SR;\n\nWhether or not a registration record (e.g. PROSPERO) or protocol for the SR was mentioned in the paper;\n\nSource of funding of the SR, classified as: ‘non-profit’, ‘for-profit’, ‘mixed’, ‘no funding’, or ‘not reported’ (for funding sources classified as ‘for-profit’ or ‘mixed’ we will record the name of the for-profit funder and classify each as ‘food industry’ or ‘other industry’);\n\nConflicts of interest of systematic reviewers as disclosed in the SR report, classified as: ‘conflict of interest present’ when at least one systematic reviewer reported a financial conflict of interest of any type, excluding current study funding or industry employment; ‘no conflict of interest’ if all systematic reviewers stated they had no conflicts; and ‘missing’ if there was no disclosure statement24; and\n\nAffiliation of the corresponding author of the SR, classified as ‘industry’ or ‘non-industry’ or ‘mixed’.\n\nData items to describe the general characteristics of the index meta-analyses. We will record the following characteristics of each of the index meta-analyses:\n\nNumber of studies included in the meta-analysis;\n\nNumber of participants included in the meta-analysis;\n\nType of population investigated (i.e. participants that were eligible for inclusion in the index meta-analysis, as specified by the systematic reviewers);\n\nType of interventions or exposures investigated;\n\nType of studies included in the meta-analysis (classified as randomized trial or non-randomized study or both; we will also classify the type of non-randomized study as specified by the systematic reviewers, e.g. ‘non-randomized trial’, ‘cohort study’, ‘case-control study’);\n\nOutcome domain (e.g. cancer mortality, weight);\n\nOutcome primacy label provided by the systematic reviewers (classified as ‘primary’, ‘secondary’ or ‘not labelled’);\n\nEffect measure (e.g. odds ratio or mean difference); and\n\nMeta-analysis model (fixed-effect, fixed-effects or random-effects).\n\nData items to evaluate selective inclusion of results in index meta-analyses. To explore whether systematic reviewers selectively included study results in meta-analyses when multiple study results were available (e.g. included data at one time point ahead of another because the former was statistically significant), we need to determine which results were eligible for inclusion in the meta-analyses. Therefore, from each SR and its corresponding SR protocol (if available), we will extract descriptions of any eligibility criteria to select effect estimates to include in the index meta-analysis. Eligibility criteria comprise lists of intervention groups, measurement instruments, time points and analyses that were eligible for inclusion (e.g. systematic reviewers state that results that were either unadjusted or adjusted for at least one prognostic factor would be included in meta-analyses). We will also extract any decision rules to select effect estimates to include in the index meta-analysis. Decision rules comprise strategies to either select one effect estimate or combine effect estimates when multiple were available (e.g. systematic reviewers state that if the effects of multiple levels of red meat intake were available, only the contrast between the highest and lowest intake would be included in the meta-analysis).\n\nWe will classify each eligibility criterion and decision rule as a strategy to handle results arising from:\n\nmultiple measurement instruments;\n\nmultiple definitions/diagnostic criteria for an event;\n\nmultiple cut-points on a continuous outcome measure;\n\nmultiple time points;\n\nmultiple intervention groups;\n\nfinal and change from baseline values;\n\nmultiple analysis samples (e.g. intention-to-treat, per-protocol and as-treated);\n\nunadjusted and covariate-adjusted analyses;\n\nperiod and paired analyses in crossover trials;\n\nmultiple information sources (e.g. journal article and trials results register);\n\noverlapping samples of participants (e.g. men only and older adults only); or\n\nanother source of multiplicity25.\n\nWe will also collect from each SR information about the study data included in the index meta-analysis. Such information will include the summary statistics for each group, and the effect estimate, measure of precision (e.g. standard error or confidence interval), and direction of the effect estimate for each included study, as displayed on the forest plot or in a table/text. We will also record whether systematic reviewers declared that study outcome data:\n\n(i) were obtained from the study investigators because the data were not reported in the study publication;\n\n(ii) required some algebraic manipulation of statistics in order to include the data in the meta-analysis (e.g. calculating a standard deviation from a 95% confidence interval of the mean);\n\n(iii) originated from a report written in a language other than English which the systematic reviewers had translated into English, or;\n\n(iv) required a method of imputation (such as imputing a missing standard deviation).\n\nWe will collect from study reports outcome data that could potentially be included in the index meta-analysis, according to the eligibility criteria and decision rules specified in the SR protocol, and with how the outcome was specified in the SR. By ‘outcome data’ we mean summary statistics (e.g. number of events, sample sizes) or an effect estimate (e.g. odds ratio) and some measure of precision (e.g. standard error, 95% confidence interval), or both if available. If no SR protocol is available, we will assume that no eligibility criteria and decision rules were pre-specified, even if some were reported in the published SR (‘worst-case scenario’ assumption), and extract all study outcome data based on how the outcome was specified in the SR. For example, a meta-analysis with the outcome ‘reduction in cardiovascular disease risk (Framingham Risk Score)’, that had no SR protocol, will have all available results for this particular outcome extracted from each study (e.g. at all time points, unadjusted and covariate-adjusted analyses, regardless of whether decision rules for these measures/analyses were stated in the published SR); however, no results for any other outcomes (e.g. using an alternative cardiovascular disease risk outcome calculated with a different algorithm) will be extracted.\n\nWhen studies of more than two groups are encountered and each comparison is eligible for inclusion in a meta-analysis, systematic reviewers need to use a method that avoids multiple counting of participants. Systematic reviewers may choose to:\n\n(i) include data from only one of the experimental intervention/exposure groups and the control group;\n\n(ii) combine the two experimental intervention/exposure groups (e.g. sum the number of events across both intervention/exposure groups for binary outcomes or calculate the mean values for both experimental intervention/exposure groups for continuous outcomes), and compare this to the control group, or;\n\n(iii) include data from each experimental intervention/exposure group as separate comparisons in the meta-analysis by dividing the sample size of the control group by the number of comparisons.\n\nIf systematic reviewers pre-defined a method to deal with multi-arm studies, we will follow that method when extracting data. If systematic reviewers did not pre-define a method to deal with multi-arm studies, and:\n\n(i) selected one of the experimental intervention/exposure groups to include in the meta-analysis, we will extract the data required to calculate effect estimates for two comparisons: (a) experimental intervention/exposure A versus control, and (b) experimental intervention/exposure B versus control;\n\n(ii) combined the two experimental intervention/exposure groups, we will extract the data required to calculate effect estimates for three comparisons: (a) experimental intervention/exposure A versus control, (b) experimental intervention/exposure B versus control, and (c) combination of intervention/exposure A and B versus control;\n\n(iii) included multiple comparisons in the meta-analysis by dividing the control group in half, we will extract the data required to calculate effect estimates for two comparisons: (a) experimental intervention A versus control, and (b) experimental intervention B versus control, where for both comparisons the control group sample size will be halved.\n\nThe three methods above will be used when dealing with three-arm studies. We will extend these methods when there are more than three arms in a study.\n\nAll study effect estimates included in mean difference meta-analyses must be in units of one particular scale, although estimates can comprise a mixture of final values and change from baseline values. In contrast, the measurement scale units of study effect estimates included in standardised mean difference (SMD) meta-analyses can vary, although it is recommended that all estimates are final values, or change from baseline values, not a mixture. Therefore, for mean difference meta-analyses we will extract only data for the particular scale included by the systematic reviewer, but extract final and change from baseline values when available. For SMD meta-analyses that included final values, we will extract final values only for any relevant measurement scale (and vice versa for SMD meta-analyses that included change from baseline values).\n\nWe will only extract study outcome data that were reported completely, defined as reporting sufficient data for inclusion in a meta-analysis (i.e. reporting of an effect estimate and a measure of precision, or summary statistics that enable calculation of these); we will not request unpublished data (e.g. missing number of events) from study authors.\n\nWe will assess the risk of bias due to missing results in each index meta-analysis using the ROB-ME (“Risk Of Bias due to Missing Evidence”) tool, introduced in the 2019 edition of the Cochrane Handbook for Systematic Reviews of Interventions12. The ROB-ME tool is the first structured approach for assessing reporting biases in meta-analyses that considers both publication bias and selective non-reporting bias. Users first consider whether results known or presumed to have been generated by study investigators are unavailable for any of the included studies (e.g. by cross-checking what was pre-specified with what was reported by study authors). They then consider whether a meta-analysis is likely to be biased because of the unavailable results in the studies identified. Finally, users consider whether qualitative signals (e.g. non-comprehensive search) and the pattern of observed results suggest that additional results are likely to be missing systematically from the meta-analysis. The tool includes signalling questions, which aim to elicit information relevant to an assessment of risk of bias. Responses to the signalling questions feed into algorithms developed to guide users of the tool to judgements about risk of bias. The possible risk-of-bias judgements are: (i) low risk of bias, (ii) some concerns, and (iii) high risk of bias.\n\nFour investigators (MJP, CMK, ZD and SM) will independently perform ROB-ME assessments on each of the index meta-analyses. We will assign the four assessors to pairs and ask them to reach consensus on their ROB-ME assessments. Any discrepancies between the responses to signalling questions and risk-of-bias judgements that cannot be resolved via discussion will be adjudicated by a third investigator (JM or LB).\n\nDescriptive analysis. We will calculate descriptive summary statistics of the general characteristics of SRs and index meta-analyses. For categorical variables, we will present frequencies and percentages. For continuous variables, we will present means (with standard deviations) and medians (with interquartile ranges). We will calculate the frequencies and percentages of SR protocols and SRs reporting the different types of eligibility criteria and decision rules to select study effect estimates. We will calculate the proportion of studies that had multiple results available for inclusion in the index meta-analyses, and quantify the number of study effect estimates that were eligible for inclusion in the index meta-analyses, and the number of eligible effect estimates per study.\n\nAnalysis of selective inclusion of results. We will follow the analyses used in a previous investigation of selective inclusion of results, as described by Page et al.13,15. We will calculate a statistic (called the ‘Potential Bias Index’ (PBI)) to quantify and test for evidence of selective inclusion. In brief, this index is based on ordering the effect estimates in each study based on their magnitude and direction of effect, and then determining the position within that order where the effect estimate included in the index meta-analysis sits. The PBI is the weighted average rank position of the selected effect estimates, where the weights are the inverse of the number of effect estimates available per study. This weighting system therefore attributes greater priority to the rank positions of effect estimates where there are a larger number of effect estimates to choose from. The expression for PBI is:\n\n\n\nwhere there are k studies, ni is the number of effect estimates in study i, and Xi is the rank of the selected effect estimate in study i. Derivation of the PBI, and a worked example, are provided in Page et al.15.\n\nThe PBI ranges from 0 to 1. For meta-analyses comparing an experimental intervention/exposure with no intervention or placebo control, the PBI will have the value 1 when the effect estimate that is most favourable to the experimental intervention/exposure is always selected for inclusion from each study. By “most favourable” we mean the effect estimate that suggested the most benefit or least harm of the intervention/exposure. Conversely, the PBI will have the value of 0 when the effect estimate that is least favourable to the experimental intervention/exposure is always selected. For meta-analyses comparing different levels or patterns of intake of the same food (e.g. wholegrain bread consumed 5 days per week versus wholegrain bread consumed once a week), we will determine from the text of the review whether the systematic reviewers hypothesised that the higher or lower category would have the most benefit or least harm, and rank study effect estimates based on their favourability to the category of consumption considered to be most beneficial/least harmful. If we cannot determine the hypothesis of the systematic reviewers, we will exclude the meta-analysis from the PBI analyses. For meta-analyses comparing different foods/diets (e.g. vegan versus vegetarian diet), we will determine from the text of the review which intervention/exposure the systematic reviewers were most interested in evaluating (which we will consider the experimental intervention/exposure), and rank the study effect estimates based on their favourability to the experimental intervention/exposure. We will also perform a sensitivity analysis excluding meta-analyses comparing different foods/diets to examine the impact on the PBI.\n\nSeveral methods for selecting effect estimates are acceptable in terms of not introducing bias, including (i) randomly selecting effect estimates, (ii) selecting effect estimates based on some clinical or methodological rationale or (iii) selecting the median effect estimate25. If systematic reviewers employed selection methods ii and iii across the studies, we expect that the distribution of the selected effect estimates would be consistent with what we would observe under purely random selection, so on average, the selected effect estimates would be at the middle rank position and the PBI would take the value of 0.5. A PBI of 0.5 therefore suggests that there is no selective inclusion of the most or least favourable effect estimates. We will run a statistical test based on the PBI that has been constructed to test whether the observed selection of effect estimates is consistent with randomness of selection15. Confidence intervals (95%) for the PBI will be obtained by bootstrap resampling26.\n\nFor meta-analyses of binary outcomes, we will express all study effect estimates in terms of odds ratios (ORs) to enable ranking of them on the same metric. For meta-analyses of continuous outcomes, we will express all study effect estimates in terms of SMDs to enable ranking of them on the same metric. In addition, we will standardise the direction of effects so that ORs below 1 or SMDs below 0 represent effects that are more favourable to the experimental intervention/exposure. We will exclude from all PBI analyses index meta-analyses that included no studies with multiplicity of effect estimates, given there is no potential for selective inclusion of results in such meta-analyses.\n\nWe will also investigate the impact of any potential selective inclusion of study effect estimates on the magnitude of the resulting meta-analytic ORs and SMDs. For each of the meta-analyses of binary outcomes, we will calculate all possible meta-analytic ORs from all combinations of available study effect estimates. When the number of possible combinations is prohibitively large to calculate all combinations (i.e. >30,000), we will generate a random sampling distribution of 5,000 meta-analytic ORs. Each meta-analytic OR will be created by randomly selecting (with equal probability) an effect estimate for inclusion from each study comparison, and meta-analysing the chosen effects. For each distribution of generated meta-analyses, we will calculate (i) the percentile rank of the index meta-analytic OR; (ii) the median of all possible meta-analytic ORs, which represents the median of a distribution where study effect estimates were not selectively included, and (iii) the difference between the index meta-analytic OR and the median meta-analytic OR. When the difference between the index and median meta-analytic OR is minimal, we will conclude that any potential selective inclusion had a limited impact on the meta-analytic effect. We will use non-parametric statistics to describe these differences. We will also synthesise these differences using a random-effects meta-analysis model, with the meta-analytic weights based on the variance of the index meta-analytic OR estimate and the between-study variability estimated using the restricted maximum likelihood estimator. The Hartung-Knapp-Sidik-Jonkman confidence interval method will be used to calculate uncertainty in the combined differences27,28. We will repeat the above analyses for each of the meta-analyses of continuous outcomes by calculating meta-analytic SMDs rather than ORs. We will also convert all SMDs to log ORs by multiplying the SMDs by π/√3 = 1.81429, and will re-run the analyses including all 50 meta-analyses of ORs.\n\nWe will conduct a fixed-effect meta-analysis of the PBI obtained in the current study with that estimated in the previous study by Page et al.13. We will synthesize estimates of the PBI using a fixed-effect meta-analysis model because the number of included studies (n=2) will be too small to adequately estimate the between-study variance. We will quantify statistical inconsistency using the I2 statistic30.\n\nWe will conduct subgroup analyses to explore whether the availability of an SR protocol or registration record, the SR being funded by the food industry, and the SR having at least one author disclosing a financial conflict of interest of any type, modifies the PBI. The confidence limits and p-value for the difference in PBI between subgroups will be constructed using bootstrap methods26. We will also fit a linear regression model to explore whether the PBI is modified by the number of available effect estimates in a study.\n\nWe will undertake a series of sensitivity analyses to investigate whether the PBI is robust to certain assumptions. For SRs without protocols or registration records, we will not be able to determine whether the eligibility criteria and decision rules to select results in the methods section of the SR were developed prior to or while undertaking the SR. Therefore, in these SRs, our primary calculation of the PBI will be based on the set of study effect estimates that were compatible with the assumption of ‘no pre-specified eligibility criteria or decision rules’. However, we will perform a sensitivity analysis where study effect estimates that were compatible only with the eligibility criteria and decision rules in the methods sections of the SR are included, to examine if this affected the PBI.\n\nIn our primary analysis of meta-analyses of continuous outcomes, we will convert all study effect estimates to SMDs to allow us to calculate the PBI in circumstances where multiple effect estimates were available for the same outcome domain, but measured on different scales. However, there is not necessarily a one-to-one relationship between the rank positions of effect estimates based on the mean difference and SMD (because the SMD additionally depends on the pooled standard deviation). Therefore, in a sensitivity analysis we will calculate the PBI based on the rank positions of the mean difference for the subset of study effect estimates that were measured on the same scale as the effect estimate included in the index meta-analysis. This will allow us to assess more accurately whether systematic reviewers had selectively included study effect estimates based on the magnitude of the mean difference in raw measurement scale units.\n\nWe anticipate that in some study reports, only an effect estimate and its standard error or 95% confidence interval will be presented (that is, the number of events or means and standard deviations per group will not be available). In this circumstance, to include the result in a meta-analysis, algebraic manipulation will be required. Algebraic manipulation may be considered challenging by some systematic reviewers, so effect estimates requiring algebraic manipulation may not have been considered by reviewers in the set of effect estimates to potentially include in the meta-analysis. For the primary calculation of the PBI, we will exclude study effect estimates that required algebraic manipulation; however, we will undertake a sensitivity analysis to explore whether the PBI is modified when we include these study effect estimates.\n\nFinally, in our primary analysis of investigating the impact of any potential selective inclusion of study effect estimates on meta-analytic effect estimates, we will use the random-effects meta-analysis model to pool effect estimates when calculating the distribution of possible meta-analytic effects. We will also perform a sensitivity analysis to explore whether our primary analysis is modified when the distribution of meta-analytic effect estimates are calculated using a fixed-effect model.\n\nAnalysis of risk of bias due to missing results. We will calculate the agreement in responses to signalling questions and risk-of-bias judgements for the ROB-ME tool for consensus assessments across the pairs of reviewers using the weighted Kappa statistic and percentage agreement (both metrics will be presented with 95% confidence intervals)31. We will interpret Kappa values as poor (≤0.00), slight (0.01–0.20), fair (0.21–0.40), moderate (0.41–0.60), substantial (0.61–0.80), or almost perfect (0.81–1.00)32.\n\nAfter reaching consensus on ROB-ME judgements across the reviewer pairs, we will calculate the frequency and percentage of index meta-analyses rated at ‘low risk of bias’, ‘high risk of bias’ or ‘some concerns’. We will conduct subgroup analyses to explore whether the SR being funded by a for-profit company, and the SR having at least one author reporting a conflict of interest of any type, were associated with the index meta-analysis being rated at high risk of bias due to missing results.\n\nWe plan to use Stata version 15 software33 to conduct all analyses.\n\nThe sample size of 50 SRs with meta-analysis was primarily selected for feasibility reasons given our available resources. This was informed by the time taken to search, screen, extract data and undertake the analysis in a previous similar study13. This sample size will allow estimation of the PBI to within a margin of error of ±0.05, assuming each meta-analysis includes an average of 8.1 studies, with 2.2 effect estimates per study (as observed in Page et al.13).\n\nWe have run the searches, piloted the screening form and started screening titles and abstracts against the eligibility criteria.\n\nDissemination of the results will be through peer-reviewed publications and presentations at conferences. We will make all data collected from this study publicly available via the Open Science Framework.\n\n\nDiscussion\n\nTo our knowledge, this is the first study to investigate bias due to selective inclusion of results and bias due to missing results in SRs with meta-analyses of the association between food/diet and health-related outcomes. Our study will address several aspects of selective inclusion of results that have not yet been explored, including the extent to which it occurs in meta-analyses of binary outcomes and in meta-analyses of non-randomized studies, and whether the practice is associated with funding source and conflicts of interest of the systematic reviewers. Our study will also provide the first evaluation of the measurement properties of the recently developed ROB-ME tool, which should provide insight into any possible revisions that may need to be made to the tool.\n\nOur study has several strengths. We have pre-specified methods to identify, select and collect data from eligible SRs and studies, and will declare any modifications to the protocol in the final report. We will use systematic methods to identify eligible SRs with meta-analyses, including use of explicit inclusion criteria, sensitive search strategies, duplicate selection and collection of data from SRs and studies, and standardised and pilot-tested data collection forms.\n\nThere are also some limitations of our planned methods. Most of the studies included in the index meta-analyses are unlikely to have been registered or have analysis plans available, which will make it challenging to detect selective non-reporting of results reliably. For example, in a cross-sectional analysis of 264 randomized trials of nutrition and dietetics interventions published in 2016, only 62 (24%) were registered prospectively34; the proportion of non-randomized nutrition studies that are prospectively registered is likely to be far lower. In addition, our investigations of whether selective inclusion of results and risk of bias due to missing results is associated with conflicts of interest of systematic reviewers relies on systematic reviewers declaring such interests in the SR report; however, previous research suggests that the level of conflict of interest disclosure in nutrition research articles is low35,36.\n\n\nConclusion\n\nMeta-analyses of nutrition research underpin the recommendations of dietary guidelines. Therefore, it is essential that the findings of such meta-analyses are robust. Our study will examine previously underexplored sources of bias in meta-analyses of nutrition research. The findings may have implications for the design, conduct and reporting of future SRs with meta-analyses of the association between food/diet and health-related outcomes.\n\n\nEthics\n\nEthics approval is not required because information will only be extracted from published studies.\n\n\nData availability\n\nNo data are associated with the article.\n\nOpen Science Framework: Investigation of Risk Of Bias due to Unreported and SelecTively included results in meta-analyses of nutrition research: the ROBUST study. https://doi.org/10.17605/OSF.IO/C827Z21\n\nThis project contains the following extended data:\n\nROBUST_protocol_appendix_1_20190920.pdf (PDF containing study search strategy)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nGBD 2017 Diet Collaborators: Health effects of dietary risks in 195 countries, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet. 2019; 393(10184): 1958–72. PubMed Abstract \| Publisher Full Text\n\nBlake P, Durão S, Naude CE, et al.: An analysis of methods used to synthesize evidence and grade recommendations in food-based dietary guidelines. Nutr Rev. 2018; 76(4): 290–300. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWhiting P, Savović J, Higgins JP, et al.: ROBIS: A new tool to assess risk of bias in systematic reviews was developed. J Clin Epidemiol. 2016; 69: 225–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPage MJ, McKenzie JE, Chau M, et al.: Methods to select results to include in meta-analyses deserve more consideration in systematic reviews. J Clin Epidemiol. 2015; 68(11): 1282–91. PubMed Abstract \| Publisher Full Text\n\nMayo-Wilson E, Fusco N, Li T, et al.: Multiple outcomes and analyses in clinical trials create challenges for interpretation and research synthesis. J Clin Epidemiol. 2017; 86: 39–50. PubMed Abstract \| Publisher Full Text\n\nPage MJ, McKenzie JE, Forbes A: Many scenarios exist for selective inclusion and reporting of results in randomized trials and systematic reviews. J Clin Epidemiol. 2013; 66(5): 524–37. PubMed Abstract \| Publisher Full Text\n\nLi T, Mayo-Wilson E, Fusco N, et al.: Caveat emptor: the combined effects of multiplicity and selective reporting. Trials. 2018; 19(1): 497. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmucker C, Schell LK, Portalupi S, et al.: Extent of non-publication in cohorts of studies approved by research ethics committees or included in trial registries. PLoS One. 2014; 9(12): e114023. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDechartres A, Atal I, Riveros C, et al.: Association Between Publication Characteristics and Treatment Effect Estimates: A Meta-epidemiologic Study. Ann Intern Med. 2018; 169(6): 385–93. PubMed Abstract \| Publisher Full Text\n\nGoldacre B, Drysdale H, Dale A, et al.: COMPare: a prospective cohort study correcting and monitoring 58 misreported trials in real time. Trials. 2019; 20(1): 118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChan AW, Hróbjartsson A, Haahr MT, et al.: Empirical evidence for selective reporting of outcomes in randomized trials: comparison of protocols to published articles. JAMA. 2004; 291(20): 2457–65. PubMed Abstract \| Publisher Full Text\n\nPage MJ, Higgins JPT, Sterne JAC: Chapter 13: Assessing risk of bias due to missing results in a synthesis. In: Higgins JPT, Thomas J, Chandler J, Cumpston M, Li T, Page MJ, et al., editors. Cochrane Handbook for Systematic Reviews of Interventions. 2nd Edition. Chichester (UK): John Wiley & Sons; 2019. Publisher Full Text\n\nPage MJ, Forbes A, Chau M, et al.: Investigation of bias in meta-analyses due to selective inclusion of trial effect estimates: empirical study. BMJ Open. 2016; 6(4): e011863. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Rezende LFM, Rey-López JP, de Sá TH, et al.: Reporting bias in the literature on the associations of health-related behaviors and statins with cardiovascular disease and all-cause mortality. PLoS Biol. 2018; 16(6): e2005761. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPage MJ, McKenzie JE, Green SE, et al.: An empirical investigation of the potential impact of selective inclusion of results in systematic reviews of interventions: study protocol. Syst Rev. 2013; 2: 21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHiggins JPT, Thomas J, Chandler J, et al.: Cochrane Handbook for Systematic Reviews of Interventions. 2nd Edition. Chichester (UK): John Wiley & Sons; 2019. Reference Source\n\nPage MJ, Shamseer L, Altman DG, et al.: Epidemiology and Reporting Characteristics of Systematic Reviews of Biomedical Research: A Cross-Sectional Study. PLoS Med. 2016; 13(5): e1002028. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhong VW, Van Horn L, Cornelis MC, et al.: Associations of Dietary Cholesterol or Egg Consumption With Incident Cardiovascular Disease and Mortality. JAMA. 2019; 321(11): 1081–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClegg A, Young J, Iliffe S, et al.: Frailty in elderly people. Lancet. 2013; 381(9868): 752–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDurão S, Kredo T, Volmink J: Validation of a search strategy to identify nutrition trials in PubMed using the relative recall method. J Clin Epidemiol. 2015; 68(6): 610–6. PubMed Abstract \| Publisher Full Text\n\nPage MJ: Investigation of Risk Of Bias due to Unreported and SelecTively included results in meta-analyses of nutrition research: the ROBUST study. 2019. http://www.doi.org/10.17605/OSF.IO/C827Z\n\nHarris PA, Taylor R, Thielke R, et al.: Research electronic data capture (REDCap)--a metadata-driven methodology and workflow process for providing translational research informatics support. J Biomed Inform. 2009; 42(2): 377–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarris PA, Taylor R, Minor BL, et al.: The REDCap consortium: Building an international community of software platform partners. J Biomed Inform. 2019; 95: 103208. PubMed Abstract \| Publisher Full Text\n\nGrundy Q, Dunn AG, Bourgeois FT, et al.: Prevalence of Disclosed Conflicts of Interest in Biomedical Research and Associations With Journal Impact Factors and Altmetric Scores. JAMA. 2018; 319(4): 408–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLópez-López JA, Page MJ, Lipsey MW, et al.: Dealing with effect size multiplicity in systematic reviews and meta-analyses. Res Synth Methods. 2018; 9(3): 336–351. PubMed Abstract \| Publisher Full Text\n\nEfron B, Tibshirani RJ: An introduction to the bootstrap. New York, NY: Chapman & Hall; 1993. Reference Source\n\nHartung J, Knapp G: A refined method for the meta-analysis of controlled clinical trials with binary outcome. Stat Med. 2001; 20(24): 3875–89. PubMed Abstract \| Publisher Full Text\n\nSidik K, Jonkman JN: A simple confidence interval for meta-analysis. Stat Med. 2002; 21(21): 3153–9. PubMed Abstract \| Publisher Full Text\n\nChinn S: A simple method for converting an odds ratio to effect size for use in meta-analysis. Stat Med. 2000; 19(22): 3127–31. PubMed Abstract \| Publisher Full Text\n\nHiggins JP, Thompson SG, Deeks JJ, et al.: Measuring inconsistency in meta-analyses. BMJ. 2003; 327(7414): 557–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHartling L, Hamm MP, Milne A, et al.: Testing the risk of bias tool showed low reliability between individual reviewers and across consensus assessments of reviewer pairs. J Clin Epidemiol. 2013; 66(9): 973–81. PubMed Abstract \| Publisher Full Text\n\nLandis JR, Koch GG: The measurement of observer agreement for categorical data. Biometrics. 1977; 33(1): 159–74. PubMed Abstract \| Publisher Full Text\n\nStataCorp: Stata Statistical Software: Release 15. College Station, TX: StataCorp LLC. 2017. Reference Source\n\nAzar M, Riehm KE, Saadat N, et al.: Evaluation of Journal Registration Policies and Prospective Registration of Randomized Clinical Trials of Nonregulated Health Care Interventions. JAMA Intern Med. 2019; 179(5): 624–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChartres N, Fabbri A, McDonald S, et al.: Association of industry ties with outcomes of studies examining the effect of wholegrain foods on cardiovascular disease and mortality: systematic review and meta-analysis. BMJ Open. 2019; 9(5): e022912. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMandrioli D, Kearns CE, Bero LA: Relationship between Research Outcomes and Risk of Bias, Study Sponsorship, and Author Financial Conflicts of Interest in Reviews of the Effects of Artificially Sweetened Beverages on Weight Outcomes: A Systematic Review of Reviews. PLoS One. 2016; 11(9): e0162198. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "55247", "date": "21 Oct 2019", "name": "Robbie C.M. van Aert", "expertise": [ "Reviewer Expertise Statistics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe reviewed manuscript is a study protocol for studying the risk of bias due to unreported and selectively included results in meta-analyses of nutrition research. I really appreciate the effort the authors put in this detailed study protocol. Below I list some major and minor comments to improve the protocol.\nMajor comments:\nIf there is no systematic review protocol available, there is assumed that there were no eligibility criteria or decision rules pre-specified. I believe this is way too restrictive, because meta-analysts will most of the time have explained what their inclusion and exclusion criteria were in the paper. This is also what is acknowledged in the proposed study protocol, but there is still decided to not use this information. Why are the systematic reviews without study protocol not included as a separate category? It would be interesting to see whether risk of bias is smallest in the systematic reviews with study protocols, larger in the ones with a clear description in the paper, and largest in the ones without a clear description in a study protocol or the paper.\n\nThere is planned to also include study reports that were eligible for inclusion in the index meta-analysis but excluded in the systematic review. Do the authors of the study protocol expect that a list is included in any systematic review with the study reports that were excluded or are they planning to redo the literature search? If the authors will be relying on a list of excluded study reports, how are they planning to deal with situations were such a list is unavailable?\n\nThe first meta-analysis is the selected meta-analysis that will be included in the study. Is it expected that the first meta-analysis is equivalent to any other meta-analysis in the systematic review? Do the authors expect that the first meta-analysis is representative for the other meta-analyses in the systematic reviews? I can imagine that the first meta-analysis is on the main effect under study of a systematic review and that the risk of bias is expected to be the largest for this meta-analysis.\n\nMinor comments:\nP.3: The authors may want to consider to change the order of the second and third paragraph of the introduction section. I believe that it is more logical to start with explaining that not all studies are usually accessible for being included in a meta-analysis and then explain that systematic reviewers themselves can also purposefully select which studies to include.\n\nP.3: An eligibility criterion of a systematic review to be included is that it can be either a randomized trial or a non-randomized study. Please consider using stratification here such that 50% of the included systematic reviews are randomized trials and 50% non-randomized studies. I believe that this may yield relevant insights as randomized trials are usually seen as of higher quality.\n\nP.8: I have a hard time understanding what the planned analysis is for studying the impact of any potential selective inclusion of study effect estimates on the magnitude of resulting meta-analytic ORs and SMDs (last paragraph, left column). Are the authors planning to compute odds ratios for each possible constellation of the 2x2 table of each individual study and then meta-analyzing these odds ratios? I do not see how this will yield insight into the effect of selective inclusion of study effect estimates on the magnitude of the meta-analytic estimate. Moreover, did the authors already write syntax/code for this? If yes, please add this to the study protocol as well.\n\nP.8: Related to my previous minor comment, I also do not understand why converting of SMDs to log ORs is necessary. Why not conducting such an analysis based on SMDs?\n\nP.8: A fixed-effect meta-analysis is planned to be used to combine the PBI that will be observed in the planned study with a PBI obtained in an earlier study. I doubt whether it is useful to meta-analyze these to PBI-values. Why not interpreting the PBI-value that will be obtained independently from the previously obtained PBI-value? It is, of course, good to relate this PBI-value to the previously obtained one, but I do not really see the need for meta-analyzing the two. Especially not since there are only two PBI-values that can be combined, so it is perfectly possible to interpret the two independently of each other. If the authors still decide to use a fixed-effect meta-analysis for combining the PBI values, please report the I2 statistic together with its confidence interval. The computed I2 statistic will be very uncertain in case of two studies and it is important to acknowledge this uncertainty.\n\nP.8: The effect of several independent variables on PBI will be tested by means of bootstrap methods. Why are not more conventional methods used for testing these independent variables such as regression analysis?\n\nP.9: The impact of any potential selective inclusion of study effect estimates on meta-analytic effect estimates will be studied using random-effects meta-analysis. Please also report what estimator will be used for estimating the between-study variance in the random-effects meta-analysis.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "5197", "date": "05 Feb 2020", "name": "Matthew Page", "role": "Author Response", "response": "Major comments:If there is no systematic review protocol available, there is assumed that there were no eligibility criteria or decision rules pre-specified. I believe this is way too restrictive, because meta-analysts will most of the time have explained what their inclusion and exclusion criteria were in the paper. This is also what is acknowledged in the proposed study protocol, but there is still decided to not use this information. Why are the systematic reviews without study protocol not included as a separate category? It would be interesting to see whether risk of bias is smallest in the systematic reviews with study protocols, larger in the ones with a clear description in the paper, and largest in the ones without a clear description in a study protocol or the paper.AUTHOR RESPONSE: In our primary analysis, we will ignore the eligibility criteria and decision rules to select results that were specified in the published SR, because there is a risk that such criteria and rules were created by systematic reviewers post-hoc. For example, systematic reviewers may have examined the study results for different scales measuring satiety, and crafted a post-hoc decision rule for scales that prioritises inclusion of the result with the largest effect estimate or smallest P value. However, as noted in paragraph eight of the section on “Analysis of selective inclusion of results”, “…we will perform a sensitivity analysis where study effect estimates that were compatible only with the eligibility criteria and decision rules in the methods sections of the SR are included, to examine if this affected the PBI”. We also state in paragraph seven of this section that “We will conduct subgroup analyses to explore whether the availability of an SR protocol or registration record…modifies the PBI”. In other words, we will compare selective inclusion in SRs with a protocol/registration entry versus SRs without. There is planned to also include study reports that were eligible for inclusion in the index meta-analysis but excluded in the systematic review. Do the authors of the study protocol expect that a list is included in any systematic review with the study reports that were excluded or are they planning to redo the literature search? If the authors will be relying on a list of excluded study reports, how are they planning to deal with situations were such a list is unavailable?AUTHOR RESPONSE: We have clarified in paragraph two of the section, “Selection of index meta-analyses”, that “We will also retrieve reports of studies that were included in the review but excluded from the meta-analysis by the systematic reviewers…)”. We expect to be able to identify which of the studies included in the review were omitted from the meta-analysis given that all (or nearly all) systematic reviews cite every study included in the review. We have added the following text to this paragraph: “We also expect that in some systematic reviews, citations to ‘near-miss’ studies along with reasons for exclusion, will be provided. For these reviews, we will scan the reasons for exclusion, and where these reasons indicate there was no useable data, we will retrieve the study to confirm that no data were available for inclusion in the review.” We will not redo the literature search. The first meta-analysis is the selected meta-analysis that will be included in the study. Is it expected that the first meta-analysis is equivalent to any other meta-analysis in the systematic review? Do the authors expect that the first meta-analysis is representative for the other meta-analyses in the systematic reviews? I can imagine that the first meta-analysis is on the main effect under study of a systematic review and that the risk of bias is expected to be the largest for this meta-analysis.AUTHOR RESPONSE: We do not expect the first meta-analysis will necessarily be representative of all other meta-analyses in the systematic review. However, the first meta-analysis is likely to be the most (or one of the most) important analysis in the review on which the conclusions of the review are based. For this reason, we have chosen to focus our assessment on the first meta-analysis. Minor comments:P.3: The authors may want to consider to change the order of the second and third paragraph of the introduction section. I believe that it is more logical to start with explaining that not all studies are usually accessible for being included in a meta-analysis and then explain that systematic reviewers themselves can also purposefully select which studies to include.AUTHOR RESPONSE: We would prefer to retain the order of the second and third paragraph, as we think it is more logical to start with explaining how meta-analyses may be biased because of actions of review authors (i.e. selective inclusion of results), followed by actions beyond review authors’ control (i.e. non-reporting by study authors). P.3: An eligibility criterion of a systematic review to be included is that it can be either a randomized trial or a non-randomized study. Please consider using stratification here such that 50% of the included systematic reviews are randomized trials and 50% non-randomized studies. I believe that this may yield relevant insights as randomized trials are usually seen as of higher quality.AUTHOR RESPONSE: As noted under “Selection of index meta-analyses”, we are already stratifying the sample so that it includes 25 meta-analyses of binary outcomes and 25 meta-analyses of continuous outcomes. The screening of titles and abstracts conducted thus far suggests there is a strong association between outcome type and type of included study in meta-analyses of nutrition research. That is, meta-analyses of continuous outcomes (such as weight) are based typically on data from randomized trials, and meta-analyses of binary outcomes (such as all-cause mortality) are based typically on data from non-randomized studies. Therefore, we believe our current stratification approach (by outcome type) will lead to a roughly equal sample of meta-analyses of randomized trials and meta-analyses of non-randomized studies. P.8: I have a hard time understanding what the planned analysis is for studying the impact of any potential selective inclusion of study effect estimates on the magnitude of resulting meta-analytic ORs and SMDs (last paragraph, left column). Are the authors planning to compute odds ratios for each possible constellation of the 2x2 table of each individual study and then meta-analyzing these odds ratios? I do not see how this will yield insight into the effect of selective inclusion of study effect estimates on the magnitude of the meta-analytic estimate. Moreover, did the authors already write syntax/code for this? If yes, please add this to the study protocol as well.AUTHOR RESPONSE: We plan to generate all possible meta-analytic effects that could be generated from the various combinations of study effect estimates available. For some studies included in an index meta-analysis, there will only have been one possible effect estimate available from the study report. For these studies, that sole effect estimate will be included in every iteration of the meta-analysis. For studies that have multiple effect estimates that are eligible for inclusion in the index meta-analysis (e.g. based on multiple scales or time points), we will randomly select a study effect estimate to include in each iteration of the meta-analysis. We will repeat this process until every possible meta-analytic effect estimate is computed (unless the number of possible combinations is prohibitively large to calculate all combinations (i.e. >30,000)). After calculating all possible meta-analytic effect estimates that the systematic reviewers could have generated, we will compare the meta-analytic effect estimate that was reported by the systematic reviewers with the median of all possible meta-analytic effect estimates that could have been generated. If the meta-analytic effect estimate presented by the systematic reviewers is much greater in magnitude than the median of all possible meta-analytic effect estimates, this suggests that systematic reviewers’ decision to selectively include study effect estimates had a considerable impact on the meta-analytic effect estimate reported. We have uploaded a worked example to the Open Science Framework (https://osf.io/pn38b/). P.8: Related to my previous minor comment, I also do not understand why converting of SMDs to log ORs is necessary. Why not conducting such an analysis based on SMDs?AUTHOR RESPONSE: To undertake the analysis including all 50 meta-analyses, we will have to use the same effect measure. Of the 50 included meta-analyses, 25 will be of continuous outcomes using the SMD, and 25 will be binary outcomes using the OR. We therefore need to either convert the SMDs to (log)ORs or the (log)ORs to SMDs. Given that we have an equal number of effect estimates to transform, we had no particular reason to adopt transforming (log)ORs to SMDs ahead of SMDs to (log)ORs. P.8: A fixed-effect meta-analysis is planned to be used to combine the PBI that will be observed in the planned study with a PBI obtained in an earlier study. I doubt whether it is useful to meta-analyze these two PBI-values. Why not interpreting the PBI-value that will be obtained independently from the previously obtained PBI-value? It is, of course, good to relate this PBI-value to the previously obtained one, but I do not really see the need for meta-analyzing the two. Especially not since there are only two PBI-values that can be combined, so it is perfectly possible to interpret the two independently of each other. If the authors still decide to use a fixed-effect meta-analysis for combining the PBI values, please report the I2 statistic together with its confidence interval. The computed I2 statistic will be very uncertain in case of two studies and it is important to acknowledge this uncertainty.AUTHOR RESPONSE: We believe there is value in meta-analysing the two PBI estimates. Doing so will yield a single estimate with greater precision. We plan to present a forest plot with estimates of PBI from the two studies, along with the combined PBI. Given there are only two estimates, we are not convinced that the I2 statistic will provide a useful quantification of inconsistency, and not more than can be obtained from visually inspecting the overlap in confidence intervals of the PBIs from each study. P.8: The effect of several independent variables on PBI will be tested by means of bootstrap methods. Why are not more conventional methods used for testing these independent variables such as regression analysis?AUTHOR RESPONSE: We plan to use bootstrap methods to estimate the confidence limits and P values since the PBI is a complex metric for which statistical theory concerning its distribution does not currently exist. We now provide more specific detail about our proposed bootstrap methods. P.9: The impact of any potential selective inclusion of study effect estimates on meta-analytic effect estimates will be studied using random-effects meta-analysis. Please also report what estimator will be used for estimating the between-study variance in the random-effects meta-analysis.AUTHOR RESPONSE: We stated in paragraph five of the section “Analysis of selective inclusion of results”, that the between-study variability will be “…estimated using the restricted maximum likelihood estimator”." } ] }, { "id": "55246", "date": "30 Oct 2019", "name": "Joshua Wallach", "expertise": [ "Reviewer Expertise Meta-research", "meta-analysis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study protocol, Page et al. outline a project focused on exploring potential biases due to selective inclusion of results and missing results in systematic reviews of food/diet-outcome relationships. In particular, the authors have 3 main objectives:\nTo explore whether systematic reviews selectively include study effect estimates in meta-analyses when multiple effects are available.\n\nTo explore what impact selective inclusion of study effect estimates may have on meta-analytic effects.\n\nTo explore the risk of bias due to missing results in meta-analyses.\n\nIn the introduction of the protocol, the authors clearly justify the rationale for, and objectives of, the study. As the authors outline, if systematic reviews are flawed, the recommendations that could inform dietary guidelines could be misleading or harmful. Nutrition/dietary exposures are difficult to evaluate in observational studies, and numerous prior empirical evaluations have suggested concerns related to various biases.\n\nThe study design outlined by the authors is appropriate. Furthermore, most of the methods are clear and are sufficient to allow replication by others. A few minor suggestions:\nIt would be helpful if the authors provided more information about their search for SRs. In particular, the authors note that they will use a PubMed to identify meta-analyses. Previous studies have outlined difficulties searching for meta-analyses. Is there a validated search strategy that the authors considered for systematic reviews/meta-analyses?\n\nOther comments:\nIn the introduction, the authors highlight that systematic reviews evaluating the association between food/diet and health-related outcomes could inform dietary guidelines. Have the authors considered limiting their sample to reviews that actually were used to inform guidelines?\n\nWhen selecting one meta-analysis, why focus on the first identified analysis? Could the authors have considered the primary outcome? Or, if the goal is identify potentially selectively reported outcomes, only focusing on those with results highlighted in the abstract?\n\nWhen looking at decision rules to select effect estimates: what if a meta-analysis includes multiple analyses based on different decision rules? It seems like this would make it difficult to evaluate selective inclusion?\n\nI am a bit confused by this statement \"If no SR protocol is available, we will assume that no eligibility criteria and decision rules were pre-specified, even if some were reported in the published SR ('worst-case scenario' assumption), and extract all study outcome data based on how the outcome was specified in the SR.\" Why not use the information in the published SR?\n\nWould it be helpful if the authors discussed the potential bias index in the introduction? This is an important part of the study, but it is not discussed until the data analysis.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "5198", "date": "05 Feb 2020", "name": "Matthew Page", "role": "Author Response", "response": "It would be helpful if the authors provided more information about their search for SRs. In particular, the authors note that they will use a PubMed to identify meta-analyses. Previous studies have outlined difficulties searching for meta-analyses. Is there a validated search strategy that the authors considered for systematic reviews/meta-analyses?AUTHOR RESPONSE: We decided to search PubMed and Epistemonikos for meta-analyses of food/diet-outcome relationships. In PubMed, we have used the following terms to identify meta-analyses: “meta-analysis[pt] OR meta-analy*[ti]”. That is, articles will need to have been classified by the PubMed indexers using the \"Meta-Analysis\" [Publication Type] MeSH term, or include the terms “meta-analysis”, “meta-analyses”, “meta-analytic” or other variants in the title. Various search filters designed to retrieve systematic reviews exist (see https://sites.google.com/a/york.ac.uk/issg-search-filters-resource/filters-to-identify-systematic-reviews). However, these are designed to retrieve systematic reviews with or without meta-analyses, and typically have high sensitivity given the lack of consensus on what constitutes a systematic review. We decided to opt for a more targeted PubMed search strategy for articles classified by database indexers or the authors themselves as meta-analyses, given we are including systematic reviews only if they present a meta-analysis. We believe this search strategy will be sufficiently comprehensive given the consensus on what constitutes a meta-analysis. The other database we are searching, Epistemonikos, has been populated by conducting systematic searches for systematic reviews (with or without meta-analyses) indexed in 10 databases (listed at https://www.epistemonikos.org/en/about_us/methods#). We have provided this additional information under “Search for SRs”. In the introduction, the authors highlight that systematic reviews evaluating the association between food/diet and health-related outcomes could inform dietary guidelines. Have the authors considered limiting their sample to reviews that actually were used to inform guidelines?AUTHOR RESPONSE: We decided not to limit our study to systematic reviews that were used to inform dietary guidelines for two reasons. First, investigators have found that most dietary guidelines are not underpinned by systematic reviews. For example, Blake et al. found that of 32 food-based dietary guidelines published from 2010 to 2016, only 10 (31%) were informed by previously published systematic reviews (https://www.ncbi.nlm.nih.gov/pubmed/29425371). Therefore, relying on dietary guidelines to identify systematic reviews may result in a smaller than desired sample. Second, the systematic reviews used to inform dietary guidelines are not likely to be current because of the time-lag between development and publication of guidelines. By limiting our focus to recently published systematic reviews (Jan 2018 to Jul 2019), we will be able to comment on current issues with transparency and risk of bias in reviews. When selecting one meta-analysis, why focus on the first identified analysis? Could the authors have considered the primary outcome? Or, if the goal is identify potentially selectively reported outcomes, only focusing on those with results highlighted in the abstract?AUTHOR RESPONSE: We have decided not to focus on the primary outcome of the systematic review because many reviews do not specify a primary review outcome. For example, Bassani et al. found that of 480 systematic reviews in dentistry indexed in PubMed in 2017, only 151 (32%) specified a primary outcome (https://www.ncbi.nlm.nih.gov/pubmed/30716451). Also, in a sample of reviews not restricted by topic that were indexed in MEDLINE in Feb 2014, 136/288 (47%) specified a primary outcome (https://www.ncbi.nlm.nih.gov/pubmed/27218655). Therefore, assuming that many recent reviews will not specify a primary outcome, we need an alternative rule to select meta-analyses that can be applied across all reviews. We have opted for the first meta-analytic result reported in the review because the first meta-analysis is likely to be the most (or one of the most) important analysis in the review on which the conclusions of the review are based. When looking at decision rules to select effect estimates: what if a meta-analysis includes multiple analyses based on different decision rules? It seems like this would make it difficult to evaluate selective inclusion?AUTHOR RESPONSE: In our previous study investigating selective inclusion of results (https://www.ncbi.nlm.nih.gov/pubmed/27121706), we found that it was always clear which decision rules applied to which meta-analysis, because the rule was applicable only to a particular outcome (e.g. authors specified a hierarchy of measurement scales for pain, and a hierarchy of scales for anxiety), or authors made it clear that the rule applied to all meta-analyses (e.g. stated that for all meta-analyses of continuous outcomes, final values were preferred over change from baseline values). We assume that the same level of specificity will occur in the systematic reviews examined in current study. I am a bit confused by this statement \"If no SR protocol is available, we will assume that no eligibility criteria and decision rules were pre-specified, even if some were reported in the published SR ('worst-case scenario' assumption), and extract all study outcome data based on how the outcome was specified in the SR.\" Why not use the information in the published SR?AUTHOR RESPONSE: In our primary analysis, we will ignore the eligibility criteria and decision rules to select results that were specified in the published SR, because there is a risk that such criteria and rules were created by systematic reviewers post-hoc. For example, systematic reviewers may have examined the study results for different scales measuring satiety, and crafted a post-hoc decision rule for scales that prioritises inclusion of the result with the largest effect estimate or smallest P value. However, as noted in paragraph eight of the section on “Analysis of selective inclusion of results”, “…we will perform a sensitivity analysis where study effect estimates that were compatible only with the eligibility criteria and decision rules in the methods sections of the SR are included, to examine if this affected the PBI”. Would it be helpful if the authors discussed the potential bias index in the introduction? This is an important part of the study, but it is not discussed until the data analysis.AUTHOR RESPONSE: We believe it is best to leave the description of the Potential Bias Index until the data analysis section, because of the extensive detail that is required to describe the index. Including information about the index in the background may detract from the rationale for the study and the objectives." } ] } ]	1	https://f1000research.com/articles/8-1760
https://f1000research.com/articles/9-72/v2	07 Feb 20	{ "type": "Opinion Article", "title": "Therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in Wuhan, China", "authors": [ "Robert L. Kruse" ], "abstract": "A novel coronavirus (2019-nCoV) originating in Wuhan, China presents a potential respiratory viral pandemic to the world population. Current efforts are focused on containment and quarantine of infected individuals. Ultimately, the outbreak could be controlled with a protective vaccine to prevent 2019-nCoV infection. While vaccine research should be pursued intensely, there exists today no therapy to treat 2019-nCoV upon infection, despite an urgent need to find options to help these patients and preclude potential death. Herein, I review the potential options to treat 2019-nCoV in patients, with an emphasis on the necessity for speed and timeliness in developing new and effective therapies in this outbreak. I consider the options of drug repurposing, developing neutralizing monoclonal antibody therapy, and an oligonucleotide strategy targeting the viral RNA genome, emphasizing the promise and pitfalls of these approaches. Finally, I advocate for the fastest strategy to develop a treatment now, which could be resistant to any mutations the virus may have in the future. The proposal is a biologic that blocks 2019-nCoV entry using a soluble version of the viral receptor, angiotensin-converting enzyme 2 (ACE2), fused to an immunoglobulin Fc domain (ACE2-Fc), providing a neutralizing antibody with maximal breath to avoid any viral escape, while also helping to recruit the immune system to build lasting immunity. The ACE2-Fc therapy would also supplement decreased ACE2 levels in the lungs during infection, thereby directly treating acute respiratory distress pathophysiology as a third mechanism of action. The sequence of the ACE2-Fc protein is provided to investigators, allowing its possible use in recombinant protein expression systems to start producing drug today to treat patients under compassionate use, while formal clinical trials are later undertaken. Such a treatment could help infected patients before a protective vaccine is developed and widely available in the coming months to year(s).", "keywords": [ "coronavirus", "Wuhan", "neutralizing antibody", "ACE2", "outbreak", "2019-nCoV" ], "content": "Introduction\n\nA mysterious illness causing pneumonia in December 2019 in Wuhan, China is now growing into a potential pandemic. These pneumonia cases were eventually characterized to be caused by a novel coronavirus (2019-nCoV)1, of which Severe Acute Respiratory Syndrome (SARS)2 and Middle East Respiratory Syndrome (MERS)3 are members. SARS and MERS famously caused their own outbreak concerns when they were originally identified. SARS caused significant economic damage to Hong Kong and Southern China, before spreading to other countries. Ultimately, SARS infected up to 8,098 people and caused 774 deaths according to the World Health Organization (WHO)4.\n\nThe novel coronavirus, 2019-nCoV, is now quickly spreading across the world after originating in Wuhan1. Human-to-human transmission of 2019-nCoV has been confirmed in familial case cluster reports5, as additional cases continue to be identified in different cities in China and countries around the world. Clinical symptoms of 2019-nCoV infection include fever, cough, and myalgia or fatigue with pneumonia demonstrated on chest CT scan imaging6. Within China, the city of Wuhan along with several others has been shut down, with individuals not allowed to leave the city in an effort to contain the virus; such efforts are largely unprecedented in a city of this size (https://www.nytimes.com/2020/01/22/world/asia/coronavirus-quarantines-history.html). For now, many travelers are being screened for fever (≥38°C) and reported recent history of travel to Wuhan in order to triage diagnostic testing7.\n\nThese efforts resemble not only what happened with SARS in 2002–2003, but also the Ebola virus outbreak in 2014–2015. During those outbreaks, special protocols were put in place to quarantine any infected individuals and identify patient contacts at risk8. Healthcare workers were also at risk, and despite extensive personal protective equipment measures, clinical providers did get infected in both outbreaks9. There were no specific, antiviral treatments for SARS or Ebola at the time of the outbreaks beyond supportive measures10,11, which is a similar situation that healthcare systems are facing with 2019-nCoV.\n\nThe dire situations facing patients in outbreak scenarios demand quick responses by the healthcare community and the biotech industry. Unfortunately, many of the traditional options that guide drug development are inadequate for outbreaks; a process that takes years can’t help patients who are dying today, and economies that are being halted. In these situations, studies have often been conducted on compassionate use, and clinical trial approvals expedited. This was most recently seen in the 2014–2015 Ebola outbreak, where a variety of clinical trial candidates were studied. Many of these therapies failed, but ultimately a vaccine did emerge that was fully protective against the virus12. It is important to note that, unlike the current situation with 2019-nCoV, Ebola had already been studied for years and this particular neutralizing vaccine made and tested in preclinical animal models years prior to the outbreak13. For 2019-nCoV, beyond knowing the sequence of spike (S) protein of the coronavirus (GenBank: MN908947.3), there are no studies on how immunogenic this particular protein will be beyond surrogate comparisons to SARS and MERS, which limits the potential ability to quickly produce a vaccine. Moreover, while a vaccine would be greatly effective in helping to stop the spread of 2019-nCoV, an effective therapy is also needed for the patients infected with 2019-nCoV today, similar to the situation of Ebola patients needing effective therapies while vaccines were being developed.\n\nIn this article, I will outline different potential treatment options that could be pursued as a therapy for 2019-nCoV virus, keeping the focus on agents that could be rapidly tested in patients today and broadly effective in spite of limited knowledge of the biology of 2019-nCoV. Simply stated, there is limited time for basic studies of 2019-nCoV in research labs, while patients need effective therapies today. I finally propose the best potential treatment option in my opinion, along with instructions on how to manufacture the therapy for testing in patients today.\n\n\nTreatment strategies against 2019-nCoV\n\nCoronavirus entry starts with the S protein binding to a target receptor on the cell surface, where after fusion is mediated at the cell membrane, delivering the viral nucleocapsid inside the cell for subsequent replication14. The S protein is famous for causing syncytial formation between infected cells and other receptor-bearing cells around them, emphasizing that the S protein does not function in just the virion state alone.\n\nA neutralizing antibody targeting the S protein on the surface of 2019-nCoV is likely the first therapy contemplated by biomedical researchers in academia and industry, providing passive immunity to disease15. The recently published genome sequence of 2019-nCoV (GenBank: MN908947.3) allows researchers to perform gene synthesis in the lab and consider expressing the S protein as an immunogen. Traditional methods of screening mice or rabbits for neutralizing antibodies may be too slow for this outbreak, but faster methods such as using phage or yeast display libraries that express antibody fragments could be used quickly to identify lead candidates for viral neutralization16,17. The challenge is that any antibody candidate would need to be rigorously tested in cell culture and animal models to confirm that it can neutralize 2019-nCoV and prevent infection. Furthermore, several isolates would need to be tested that are circulating in the population to try to assess if sufficient breadth of coverage is obtained with the neutralizing antibody. Information from other coronaviruses species like SARS would be helpful as to where to target the best epitope in order to produce neutralizing antibodies (the receptor-binding domain in the S protein is a key target)18, but again this is a slow and challenging process, which may not yield significant gains for several months. Moreover, ultimately a cocktail of antibodies may be required to ensure full protection for patients, which would add additional complexity for formulation and manufacturing. Like some of the therapeutic options discussed below, the ability to express any lead candidates in lower organisms for protein expression (bacteria, yeast, insect cells) would facilitate faster production of therapy for patients19.\n\nAn alternative strategy of generating neutralizing antibodies against 2019-nCoV S protein would be to immunize large animals (sheep, goat, cow) with the 2019-nCoV S protein, and then purifying polyclonal antibodies from the animals20. This strategy may serve an expedited service in the setting of an outbreak and has many advantages such as simplifying production and manufacturing, but has limited guarantees that each animal would produce neutralizing antisera, or what the antibody titer would be in each animal21. Moreover, there is also the human immune response against foreign immunoglobulins to other species, which would potentially complicate any treatment scenarios22. In a truly desperate scenario, this strategy may be viable for a short-term, but would not easily scale in the 2019-nCoV outbreak, which is already rapidly multiplying.\n\nBeyond targeting the surface proteins of 2019-nCoV, one could also target the RNA genome itself for degradation. This RNA genome sequence of 2019-nCoV was recently published (GenBank: MN908947.3), and one strategy that could be considered then, is the use of small interfering RNA (siRNA) or antisense oligonucleotides (ASO) to combat the virus by targeting its RNA genome23. The challenge with this strategy is multi-fold. First, the conserved RNA sequence domains of CoV-2019 are not known. Identifying conserved sequences is essential in order to optimize siRNA targeting and avoid viral escape of the oligonucleotide strategy. One could look at genome homology of 2019-nCoV to the SARS virus for comparison of conserved sequences, but this would still be guesswork. A second challenge is how the oligonucleotides would be delivered into the lungs. Advances have been made into delivery vehicles such as lipid nanoparticles that can mediate some delivery into the lungs24. It is unknown, however, if enough siRNA’s or ASO’s would be effectively delivered within the lungs to stop the infection or make a difference in its clinical course. For example, if 25% of alveolar epithelial cells in the lung had siRNA or ASO in them, that efficiency might be a great success for traditional gene therapy, but would hardly make any difference in a viral infection. Such an explanation is also likely why siRNA candidates against Ebola failed in trials25, despite significant success in preclinical animal models26,27. Lastly, even if one assumed that siRNA was effective clinically, there is a limited ability to scale up manufacturing of siRNA drugs to a large infected population. Current siRNA and ASO therapies are manufactured for rare diseases, and there are no available resources existing to manufacture the medications quickly.\n\nIdeal agents to fight 2019-nCoV would be approved small molecule drugs that could inhibit different aspects of the viral life cycle, ultimately inhibiting replication. Two classes of potential targets are viral polymerases28 and protease inhibitors29, both of which are components of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) antiviral regimens. Pilot clinical studies are already ensuing by desperate clinicians with various repurposed antiviral medicines. This has been done in every viral outbreak previously with limited success, outside of case reports30. Indeed, during the Ebola outbreak, none of the repurposed small molecule drugs were definitively shown to improve the clinical course across all patients31. The 2019-nCoV could be different, and there are initial positive reports that lopinavir and ritonavir, which are HIV protease inhibitors, have some clinical efficacy against 2019-nCoV, similar to prior studies using them against SARS32. Research should continue to be undertaken to screen other clinically available antivirals in cell culture models of 2019-nCoV, in hopes that a drug candidate would emerge useful against the virus that could be rapidly implemented in the clinic. One promising example could be remdesivir, which interferes with the viral polymerase and has shown efficacy against MERS in mouse models33. For further information, reviews of previous drug repurposing efforts for coronaviruses are provided34,35. Though these repurposed medications may hold promise, it is still reasonable to pursue novel, 2019-nCoV specific therapies to complement potential repurposed drug candidates.\n\nA simple but potentially very effective tool that can be used in infectious outbreaks is to use the serum of patients who have recovered from the virus to treat patients who contract the virus in the future36. Patients with resolved viral infection will develop a polyclonal antibody immune response to different viral antigens of 2019-nCoV. Some of these polyclonal antibodies will likely neutralize the virus and prevent new rounds of infection, and the patients with resolved infection should produce 2019-nCoV antibodies in high titer.\n\nPatients with resolved cases of 2019-nCoV can simply donate plasma, and then this plasma can be transfused into infected patients37. Given that plasma donation is well established, and the transfusion of plasma is also routine medical care, this proposal does not need any new science or medical approvals in order to be put into place. Indeed, the same rationale was used in the treatment of several Ebola patients with convalescent serum during the outbreak in 2014–2015, including two American healthcare workers who became infected38.\n\nAs the outbreak continues, more patients who survived infection will become available to serve as donors to make antisera for 2019-nCoV, and a sizeable stock of antisera could be developed to serve as a treatment for the sickest patients. Unfortunately, the exponential growth of the outbreak would work against this strategy, since the growing number of cases would likely outstrip the ability of previous patients to provide donor plasma as treatment. Moreover, convalescent patient sera would have significant variability in the potency of antiviral effect, making it less ideal37. While transfusion medicine services should certainly pursue convalescent patient sera as an option right now for patient treatment, it is ultimately limited in its effective scope of controlling the outbreak.\n\n\nProposal for new 2019-nCoV therapies\n\nThe simplest and most direct approach to combating 2019-nCoV during the outbreak would be one to neutralize the virus from entering cells, the function that antibodies normally perform in the body39. For the reasons mentioned above when discussing neutralizing antibodies, it will be difficult to validate a broadly neutralizing antibody quickly, and a challenge to make sure that the mutating RNA virus will not escape its neutralization. A cocktail antibody approach could be undertaken as was explored to treat the Ebola pandemic40, but would add complexity to the manufacturing process.\n\nHowever, there is another strategy to pursue in this scenario that does not rely on targeting the viral glycoprotein directly. In this strategy, a neutralizing effect could be obtained by targeting the viral receptor protein on the cell surface, thereby blocking the virus from binding to it and gaining entry. Fortunately, scientists have already uncovered the identity of the viral receptor in cell culture. A recent pre-print publication found that the 2019-nCoV uses the angiotensin-converting enzyme 2 (ACE2) as a receptor for cell entry41, which is the same receptor that the SARS coronavirus uses for entry42. For both viruses, the coronavirus binds to ACE2 through its S protein on the virion, where after fusion of the viral membrane and cell membrane will occur. Subsequently, the RNA virus will replicate its genome inside the cell, and ultimately make new virions that will be secreted to infect other cells. The coincidence of SARS and 2019-nCoV using ACE2 receptor opens up the possibility of using the extensive research studied on SARS entry and applying it to 2019-nCoV. Based on the SARS literature, several potential blocking strategies could be considered, which were shown to be effective in preventing infection in SARS models.\n\nThe first strategy would consist of administering to patients an agent that would bind to ACE2. The key advantage here is that the host ACE2 protein will not change, so there is no concern about escape from binding the therapeutic agent. Moreover, the virus will not have the ability to mutate and bind an entirely new host receptor in the time frame of this outbreak; such functional relationships are established by evolution over long periods. By analogy, the influenza virus changes the mutations on its surface to escape antibody neutralization every year, but it always focuses on using sialic acid on the cell surface as an entry receptor43.\n\nThere are two known options for agents to bind to ACE2. The first is using the small receptor-binding domain (RBD) from the SARS S protein that has been shown to be the key domain that binds the ACE2 receptor during entry44. Administration of this domain, 193 amino acids in size, has been shown to effectively block the entry of SARS in cell culture44. It is well within reason that SARS RBD could be given to patients, thereby binding their ACE2 proteins on target cells, preventing infection (Figure 1). There is also the potential for the equivalent RBD of 2019-nCoV to be produced and used as a therapy as well. This strategy assumes SARS and 2019-nCoV share the same binding site on ACE2, which is highly likely given the similar ACE2 binding sites of SARS and NL63 coronavirus The small size of the therapy, similar in size in nanobody domains from camelid antibodies, would enhance the perfusion of the biologic into tissues to more effectively bind to viral entry receptors45. In regards to the outbreak situation that is ongoing, the small protein facilitates the rapid production of the therapy in bacteria potentially, which would help production yields19. Moreover, bacterial production would allow RBD proteins to be produced in a wide range of production facilities today in China, which already has numerous contract research organization operations46. Alternatively, the RBD protein could be attached to an Fc fragment for extended circulation, which was done for an equivalent 212 amino acid domain from MERS. The MERS RBD-Fc fusion demonstrated the ability to block viral infection toward cell receptors, as well as to stimulate an immune response against that specific viral domain in mice47. Of note, since the RBD-Fc fusion would bind to normal cells, one would want to eliminate cytotoxic Fc domain functions through mutations that eliminate Fc receptor binding48.\n\nTarget cells expressing ACE2 include lung and gastrointestinal tissues in the human body. The large spike protein on the surface of the coronavirus binds to ACE2 on infected cells, leading to cell entry. Three proposed strategies would block this interaction would abrogate infection. In the first, the receptor-binding domain (RBD) of the spike protein from SARS or 2019-nCoV would be administered, thereby binding ACE2 and saturating available sites. Alternatively, an antibody or single chain antibody fragment (scFv) could be administered against ACE2 to accomplish the same. A third strategy would target the coronavirus virions directly by using the ACE2 extracellular domain as bait to bind to spike protein. An Fc domain fused to ACE2 would facilitate prolonged circulation of the biologic (ACE2-Fc).\n\nA second, similar strategy would be to administer an antibody that would bind to ACE2 protein, thereby preventing 2019-nCoV infection (Figure 1). This strategy was shown to effectively block SARS entry and replication in experiments42. While no ACE2 antibody sequences are published in literature indexes, monoclonal antibodies do exist and the associated hybridoma sequences could be cloned in a matter of days. There would be no concern for any viral escape from an ACE2 binding antibody, which is an advantage over neutralizing approaches against the S protein. There are a couple of design considerations when thinking about how to employ the ACE2 antibody strategy. Any effector functions would need to be removed from the Fc domain49, such that inflammation would not be caused in different tissues expressing ACE2. This would retain the long-half life endowed by the Fc domain without any of the side effects. The downside of including the Fc domain is the need to use a more expensive mammalian cell production system to preserve proper glycosylation, which would decrease the turnaround time for getting the drug to patients in the outbreak scenario. Alternatively, one could just administer a single chain variable fragment (scFv) that binds to ACE2. A nanobody or VHH domains from camelids are another option as well50,51. These could be produced in bacteria, and its small size would allow for rapid permeation into different tissues. The downside is the shorter half-life of these molecules without the Fc domain.\n\nThere are several limitations to these two options. Regarding the SARS RBD strategy, the body would likely develop an immune response to the SARS protein eventually, although the key intervention period of infection to combat 2019-nCoV would fall under this window of time, where after an immune response for both viruses would develop. Alternatively, if one were to use the homologous RBD from 2019-nCoV itself, this immune response would likely be very advantageous since it could yield both a blocking effect and a vaccination effect52. For both strategies, the dose that would be needed to block ACE2 receptors in the body across different organs is unknown, and as is the percentage of ACE2 receptors that would need to be saturated in order to slow the infection. The number of ACE2 receptors in the body, which are found in lung and gastrointestinal organs along with vascular endothelial cells among other tissues53, could ultimately prove prohibitive for this strategy. Moreover, the turnover of the ACE2 receptor on the cell surface would also influence how often the therapeutic protein would need to be administered. To solve this issue, one could increase the concentration of anti-ACE2 therapy at the crucial site of infection in the lungs, via local administration to lungs via nebulization. Lastly, there is the possibility that binding ACE2 directly could paradoxically worsen lung physiology and clinical symptoms. A study found that a fusion protein of SARS RBD to Fc domain bound ACE2 in murine lung tissue after administration, exacerbating alveolar edema via ACE2 interaction, which normally counteracts acute lung injury54. This suggests that if one were use an ACE2 binding strategy, it would be best employed early during infection or as a prophylaxis to block the initial viral infection. Ultimately, clinical trials in patients would need to investigate these potential issues.\n\nA potentially more promising strategy would be to create an antibody-like molecule that would bind to the coronavirus itself, rather than shielding cells from being infected. For this strategy, it is proposed to use a soluble version of the ACE2 receptor that would bind to the S protein of 2019-nCoV thereby neutralizing the virus (Figure 1). Again, the research on the SARS virus suggests this strategy is potentially promising. Soluble ACE2 receptor was demonstrated to block the SARS virus from infecting cells in culture42. The reported affinity of soluble ACE2 for the SARS S protein was 1.70 nM, which is comparable to the affinities of monoclonal antibodies55; it is likely that 2019-nCoV has similar affinity for ACE2. In order to use ACE2 as a therapy to treat patients, it would be advisable to convert soluble ACE2 into an immunoadhesin format fused to an immunoglobulin Fc domain (ACE2-Fc), thereby extending the lifespan of the circulating molecule, while also recruiting effector functions of the immune system against the virus. While not tested in an animal model, a previous study demonstrated that an ACE2 extracellular domain fused to the human IgG1 domain (ACE2-NN-Ig) was effective in neutralizing SARS coronavirus in vitro, with a 50% inhibitory concentration of 2 nM56. This study provides evidence then that ACE2-Fc could similarly inhibit 2019-nCoV in vitro and potentially in patients.\n\nAn additional advantage of using ACE2 as a 2019-nCoV S protein neutralizing agent is that ACE2 administration could also directly treat the pneumonia pathophysiology. A portion of patients with SARS and 2019-nCoV infection develop pneumonia, which is characterized by pulmonary edema and acute respiratory distress syndrome (ARDS)1,2. The viruses may, in part, cause ARDS through viral-induced ACE2 protein shedding and ACE2 protein decreased expression, both of which are mediated by S protein binding54. Administration of recombinant ACE2 protein has been shown to improve acute lung injury through decreasing angiotensin II levels and the hormones subsequent binding to angiotensin II type 1a receptor57. Recombinant ACE2 can also reduce ARDS in respiratory syncytial virus58 and H5N1 influenza59 infection models. Based on these promising preclinical studies, recombinant human ACE2 (rhACE2) was moved into clinical trials in order to treat ARDS in critically ill patients. A phase I trial demonstrated rhACE2 was well tolerated with no effects seen on the cardiovascular system60. A phase II trial demonstrated on-target efficacy in reducing Ang1-8 peptide levels, but did not show significant modulation of respiratory parameters61. It remains to be seen whether rhACE2 administration has the same clinical benefits in treating ARDS that have been seen in animal models, and whether ACE2-Fc administration could alleviate ARDS in 2019-nCoV patients.\n\nThe proposed therapy for 2019-nCoV patients would consist of the extracellular domain of the ACE2 protein fused to a human immunoglobulin G Fc domain (Figure 2A). Studies have shown that the ACE2 amino acids 18 – 615 appear to be sufficient for SARS S protein binding62, which also covers the peptidase domain necessary for ACE2 enzymatic function. It is possible a smaller portion of the extracellular ACE2 domain would be adequate for S protein binding, although a smaller version would lack enzyme activity beneficial in treating lung injury. Further studies are needed to define the minimal ACE2 domain necessary for 2019-nCoV S protein binding to construct even smaller ACE2-Fc proteins. While we do not know the structure of the 2019-nCoV S protein or how it binds to the ACE2 receptor yet, it is reasonable for now to assume that the same ACE2 protein domains utilized by the SARS virus are also bound by 2019-nCoV to infect cells.\n\n(A) The extracellular domain of ACE2 is appended onto the human immunoglobulin Fc domain, including the hinge region. The Fc domain facilitates dimerization of two ACE2 domains. (B) The amino acid sequence of the ACE2-Fc fusion protein is provided. The ACE2 domain consists of amino acids 18–615 of the human ACE2 protein (blue; UniProtKB - Q9BYF1). The sequence of the human immunoglobulin G isotype 1 constant region is provided (green; UniProtKB - P01857). A secretion signal from a human immunoglobin heavy chain is provided (red; UniProtKB - A0A0C4DH39).\n\nThe advantage of the Fc domain is endowing a longer-half life of the drug, which could enable healthcare workers to potentially be given drug doses prophylactically before seeing infected patients. Indeed, the half-life of recombinant ACE2 was extended from less than two hours to over one week in mice when formatted as a recombinant ACE2-Fc therapy in a study evaluating treatment for hypertension63. One difference from the prior blocking agent strategies is that the effector functions of the Fc domain could be retained in this molecule, allowing recruitment of dendritic cells, macrophages, and natural killer cells through the CD16 receptor against viral particles or infected cells. This may facilitate faster activation of the host antiviral immune response and elimination of the virus, which was illustrated in a SARS mouse model where Fc engaging antibodies were more potent in eliminating SARS via activation of phagocytic cells compared to antibodies that neutralized virus alone64. Overall, the ACE2-Fc fusion protein would have many of the same benefits of a traditional neutralizing antibody that would be sought as a treatment for the infection, but represent one with maximal breadth and potency since the 2019-nCoV could not escape its neutralization, given the same protein is also its receptor for cell entry. Indeed, it has been shown that the pathogenicity of SARS versus the more mild human coronavirus NL63 was related to a lower affinity of NL63 for human ACE2 versus SARS, with NL63 S protein reducing ACE2 levels less than SARS S protein65. Therefore, if 2019-nCoV were to try to escape ACE2 neutralization via decreasing affinity, it would mutate into a less pathogenic virus. This is similar to the re-emergent SARS virus in 2003-2004, which had lower affinity for human ACE2 and resulted in less severe infection and no secondary transmission66. Thus, 2019-nCoV could be presented with an evolutionary trap when faced with potential ACE2-Fc therapy, leading toward a more benign clinical course.\n\nTo give some additional support to the potential of a receptor-immunoadhesin being a potential antiviral strategy, it should be noted that CD4-Fc or CD4-IgG was one of the early agents developed as a potential HIV medication67. The protein contained the first two domains of the CD4 receptor that are known to bind gp120 on the surface of infected HIV cells. CD4-IgG was shown to neutralize HIV in vitro, preventing infection. The protein was also safe when administered in patients, although only limited-to-mild clinical benefit was achieved68,69. Updated enhanced versions of CD4-IgG have been developed that additionally have a small peptide derived from the co-receptor, CCR5, enhancing affinity and giving even more potent neutralizing activity, essentially 100% of HIV isolates and making rhesus macaques resistant to multiple simian-human immunodeficiency virus challenges70,71. While HIV and 2019-nCoV are very different viruses, with different cell types, kinetics, and clinical courses, the previous results with HIV are encouraging that this could be a therapeutic strategy for 2019-nCoV. If anything, 2019-nCoV is likely more amenable to this neutralizing therapy given that the respiratory virus will only cause an acute infection, unlike HIV, which causes chronic infection in hosts with different cellular reservoirs.\n\nOne potential limitation of the ACE2-Fc strategy is that the increase in levels of extracellular ACE2 could have unknown effects on the body, particularly when elevated for a prolonged time via Fc domain extended half-life. Small levels of extracellular ACE2 are already secreted by tissues, so the circulation of this extracellular domain would not be unprecedented72. Moreover, recombinant ACE2 protein was well-tolerated by healthy patients in a phase I trial, and by patients with lung injury in a phase II trial, suggesting treating 2019-nCoV patients with ACE2-Fc will also tolerated. If investigators are still concerned, critical amino acid(s) for ACE2 peptidase activity could be mutated to abolish the native function of this sequence, while retaining high affinity binding for SARS and 2019-nCoV S protein. Indeed, this possibility was previously investigated in generating an ACE2 and IgG1 fusion protein, which showed that mutation of histidine residues at position 374 and 378 of the ACE2 extracellular domain abolished peptidase activity, but retained high affinity binding to SARS S protein56. Of course, ACE2 peptidase mutation would eliminate the beneficial effects from the recombinant protein delivery of ACE2 in treating lung injury, so it is recommended that retaining ACE2 enzyme activity be pursued first. Another potential concern is that receptor binding via an antibody format could inadvertently direct 2019-nCoV toward infecting Fc receptor (CD16) positive cells, which has been shown in vitro for neutralizing antibodies in MERS73. It’s unclear what clinical significance this would have, and to what extent this would happen in vivo. Ultimately, clinical trials will be needed to delineate any specific side effects of ACE2-Fc treatment.\n\n\nAction plan and discussion\n\nThe chief objective of global health efforts against 2019-nCoV remains to effectively quarantine patients and screen individuals who may be infected to limit spread. That objective should continue going forward. What is proposed here is an option to at least give infected patients a medication quickly to help alleviate symptoms and prevent death, while vaccine efforts for 2019-nCoV continue. This could be enabled through ACE2-Fc providing a triple mechanism of action: (1) Treatment of ACE2 deficiency and lung injury, (2) virus neutralization, and (3) immune effector recruitment. Beyond infected patients, ACE2-Fc could provide passive immunity to healthcare works at risk as another benefit. Going forward, it is recommended that physicians, scientists, and biotech industry in China and elsewhere pursue manufacturing an ACE2-Fc biologic agent right now, which can immediately advance into trials. A variety of different protein expression platforms (CHO, insect, yeast) could be utilized, depending on the particular contract manufacturer’s expertise. Gene therapy could even be considered to make ACE2-Fc from a DNA or mRNA platform, but would have additional risk of uncertain delivery strategies and ultimately may slow down progress toward treating patients.\n\nThe goal would be that ACE2-Fc could treat infection in current patients preventing significant morbidities and death, while also serving as a potential prophylactic to give passive immunity to clinical providers on the frontlines, as well as individuals who may have been exposed to the virus. Essentially, ACE2-Fc could be the potent neutralizing antibody that the global health community needs to combat 2019-nCoV, today, while also treating the underlying ARDS pathophysiology causing patient mortality. It could be scaled much more quickly than convalescent patient sera, which would be dependent on infected individuals to make. ACE2-Fc would be resistant to viral escape as well, unlike potential neutralizing monoclonal antibodies that may be developed in the coming weeks to months.\n\nWhile a therapeutic strategy is being outlined here, the long-term goal of 2019-nCoV research would remain developing an effective vaccine to yield neutralizing antibodies, likely based on the S protein and specifically, the RBD protein. Such trials should happen as soon as possible, but may prove to be challenging to get the right level of immunogenicity, antigen presentation, adjuvant addition, and potent antibody stimulation. The virus could continue mutating, foiling different efforts to stimulate protective immunity. By comparison, 2019-nCoV cannot escape the ACE2-Fc treatment strategy, since it leverages its own cognate receptor for infection. As mentioned above, should 2019-nCoV attempt to escape this therapy via reduced ACE2 affinity binding, it would likely become less pathogenic, similar to the comparison of SARS versus human coronavirus NL6365. Lastly, scaling the dose of any effective vaccine would also prove to be challenging depending on the vector format (e.g. viral vector versus mRNA versus protein), and even a fully protective vaccine would not help patients who are currently infected with the virus.\n\nIn an effort to help aide researchers and industry in China to combat 2019-nCoV, the protein sequence of the ACE2-Fc construct is provided (Figure 2B). Different human Fc domains (IgG1, IgG2, IgG3, or IgG4) could be contemplated, although IgG1 traditionally has the most potency for triggering anti-microbial responses49. Similarly, an ACE2-Fc biologic without active ACE2 peptidase function could be explored as well. Given that gene synthesis of this sequence could happen within a week, the gene could be placed within the protein expression platform of choice shortly thereafter leading to protein production quickly. The availability of protein A columns and other techniques in the industry to purify antibodies would facilitate ACE2-Fc to quickly be repurposed on existing antibody manufacturing infrastructure existing in China.\n\nA final benefit of pursuing ACE2-Fc is that it could effectively be used as a therapeutic drug stockpile for future outbreaks of SARS and 2019-nCoV, and any new coronavirus that emerges from a zoonotic reservoir in the future that uses the ACE2 receptor for entry. Moreover, coronaviruses that replicate in animals across China and other countries could be studied in order to assess their entry mechanisms. By understanding entry in these other animals, one could effectively predict a receptor that could be utilized in any zoonotic transmission event, and build a new receptor immunoadhesin molecule in the future. As an example, a similar immunoadhesin, DPP4-Fc, could be envisioned for MERS based on the viral receptor, DPP4, used by that virus74. Beyond coronaviruses, this strategy could be utilized for other viruses where the risk of outbreak potential is high. ACE2-Fc could also find use in treating ARDS for other unrelated viruses and causes of acute lung injury, building on the previous clinical trial work60,61.\n\nIn summary, ACE2-Fc has the potential to be the neutralizing antibody that healthcare workers need to treat and prevent 2019-nCoV infection today and could play an important role in the cessation of the outbreak if manufacturing based on an available sequence starts soon. An alternative 2019-nCoV RBD-Fc fusion could also be pursued, if one desired the dual function of receptor blocking and vaccination in one molecule.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nZhu N, Zhang D, Wang W, et al.: A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med. 2020; NEJMoa2001017. PubMed Abstract \| Publisher Full Text\n\nKsiazek TG, Erdman D, Goldsmith CS, et al.: A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med. 2003; 348(20): 1953–1966. PubMed Abstract \| Publisher Full Text\n\nZaki AM, van Boheemen S, Bestebroer TM, et al.: Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med. 2012; 367(19): 1814–1820. PubMed Abstract \| Publisher Full Text\n\nStadler K, Masignani V, Eickmann M, et al.: SARS--beginning to understand a new virus. Nat Rev Microbiol. 2003; 1(3): 209–218. PubMed Abstract \| Publisher Full Text\n\nChan JF, Yuan S, Kok KH, et al.: A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. Lancet. 2020; pii: S0140-6736(20)30154-9. PubMed Abstract \| Publisher Full Text\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; pii: S0140-6736(20)30183-5. PubMed Abstract \| Publisher Full Text\n\nCheng VCC, Wong SC, To KKW, et al.: Preparedness and proactive infection control measures against the emerging Wuhan coronavirus pneumonia in China. J Hosp Infect. 2020; pii: S0195-6701(20)30034-7. PubMed Abstract \| Publisher Full Text\n\nSvoboda T, Henry B, Shulman L, et al.: Public health measures to control the spread of the severe acute respiratory syndrome during the outbreak in Toronto. N Engl J Med. 2004; 350(23): 2352–2361. PubMed Abstract \| Publisher Full Text\n\nFischer WA 2nd, Weber D, Wohl DA: Personal Protective Equipment: Protecting Health Care Providers in an Ebola Outbreak. Clin Ther. 2015; 37(11): 2402–2410. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAvendano M, Derkach P, Swan S: Clinical course and management of SARS in health care workers in Toronto: a case series. CMAJ. 2003; 168(13): 1649–1660. PubMed Abstract \| Free Full Text\n\nUyeki TM, Mehta AK, Davey RT Jr, et al.: Clinical Management of Ebola Virus Disease in the United States and Europe. N Engl J Med. 2016; 374(7): 636–646. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHenao-Restrepo AM, Camacho A, Longini IM, et al.: Efficacy and effectiveness of an rVSV-vectored vaccine in preventing Ebola virus disease: final results from the Guinea ring vaccination, open-label, cluster-randomised trial (Ebola Ça Suffit!). Lancet. 2017; 389(10068): 505–518. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGeisbert TW, Daddario-Dicaprio KM, Lewis MG, et al.: Vesicular stomatitis virus-based ebola vaccine is well-tolerated and protects immunocompromised nonhuman primates. PLoS Pathog. 2008; 4(11): e1000225. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTortorici MA, Veesler D: Structural insights into coronavirus entry. Adv Virus Res. 2019; 105: 93–116. PubMed Abstract \| Publisher Full Text\n\nCasadevall A, Pirofski LA: The Ebola epidemic crystallizes the potential of passive antibody therapy for infectious diseases. PLoS Pathog. 2015; 11(4): e1004717. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShin YW, Chang KH, Hong GW, et al.: Selection of Vaccinia Virus-Neutralizing Antibody from a Phage-Display Human-Antibody Library. J Microbiol Biotechnol. 2019; 29(4): 651–657. PubMed Abstract \| Publisher Full Text\n\nKeck ZY, Wang Y, Lau P, et al.: Isolation of HCV Neutralizing Antibodies by Yeast Display. Methods Mol Biol. 2019; 1911: 395–419. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElshabrawy HA, Coughlin MM, Baker SC, et al.: Human monoclonal antibodies against highly conserved HR1 and HR2 domains of the SARS-CoV spike protein are more broadly neutralizing. PLoS One. 2012; 7(11): e50366. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTripathi NK, Shrivastava A: Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development. Front Bioeng Biotechnol. 2019; 7: 420. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmidt R, Beltzig LC, Sawatsky B, et al.: Generation of therapeutic antisera for emerging viral infections. NPJ Vaccines. 2018; 3: 42–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJahrling PB, Geisbert J, Swearengen JR, et al.: Passive immunization of Ebola virus-infected cynomolgus monkeys with immunoglobulin from hyperimmune horses. Arch Virol Suppl. 1996; 11: 135–140. PubMed Abstract \| Publisher Full Text\n\nGoto M, Kuribayashi K, Umemori Y, et al.: High prevalence of human anti-mouse antibodies in the serum of colorectal cancer patients. Anticancer Res. 2010; 30(10): 4353–4356. PubMed Abstract\n\nQureshi A, Tantray VG, Kirmani AR, et al.: A review on current status of antiviral siRNA. Rev Med Virol. 2018; 28(4): e1976. PubMed Abstract \| Publisher Full Text\n\nYoungren-Ortiz SR, Gandhi NS, España-Serrano L, et al.: Aerosol Delivery of siRNA to the Lungs. Part 1: Rationale for Gene Delivery Systems. Kona. 2016; 33: 63–85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDunning J, Sahr F, Rojek A, et al.: Experimental Treatment of Ebola Virus Disease with TKM-130803: A Single-Arm Phase 2 Clinical Trial. PLoS Med. 2016; 13(4): e1001997. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThi EP, Mire CE, Lee AC, et al.: Lipid nanoparticle siRNA treatment of Ebola-virus-Makona-infected nonhuman primates. Nature. 2015; 521(7552): 362–365. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThi EP, Lee AC, Geisbert JB, et al.: Rescue of non-human primates from advanced Sudan ebolavirus infection with lipid encapsulated siRNA. Nat Microbiol. 2016; 1(10): 16142. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsai CH, Lee PY, Stollar V, et al.: Antiviral therapy targeting viral polymerase. Curr Pharm Des. 2006; 12(11): 1339–1355. PubMed Abstract \| Publisher Full Text\n\nAnderson J, Schiffer C, Lee SK, et al.: Viral protease inhibitors. Handb Exp Pharmacol. 2009; 189: 85–110. PubMed Abstract \| Publisher Full Text\n\nLitterman N, Lipinski C, Ekins S: Small molecules with antiviral activity against the Ebola virus [version 1; peer review: 2 approved]. F1000Res. 2015; 4: 38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDodd LE, Follmann D, Proschan M, et al.: A meta-analysis of clinical studies conducted during the West Africa Ebola virus disease outbreak confirms the need for randomized control groups. Sci Transl Med. 2019; 11(520): pii: eaaw1049. PubMed Abstract \| Publisher Full Text\n\nChu CM, Cheng VC, Hung IF, et al.: Role of lopinavir/ritonavir in the treatment of SARS: initial virological and clinical findings. Thorax. 2004; 59(3): 252–256. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSheahan TP, Sims AC, Leist SR, et al.: Comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against MERS-CoV. Nat Commun. 2020; 11(1): 222. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi CC, Wang XJ, Wang HR: Repurposing host-based therapeutics to control coronavirus and influenza virus. Drug Discov Today. 2019; 24(3): 726–736. PubMed Abstract \| Publisher Full Text\n\nDyall J, Coleman CM, Hart BJ, et al.: Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection. Antimicrob Agents Chemother. 2014; 58(8): 4885–4893. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMire CE, Geisbert JB, Agans KN, et al.: Passive Immunotherapy: Assessment of Convalescent Serum Against Ebola Virus Makona Infection in Nonhuman Primates. J Infect Dis. 2016; 214(suppl 3): S367–S374. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarano G, Vaglio S, Pupella S, et al.: Convalescent plasma: new evidence for an old therapeutic tool? Blood Transfus. 2016; 14(2): 152–157. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKraft CS, Hewlett AL, Koepsell S, et al.: The Use of TKM-100802 and Convalescent Plasma in 2 Patients With Ebola Virus Disease in the United States. Clin Infect Dis. 2015; 61(4): 496–502. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWalker LM, Burton DR: Passive immunotherapy of viral infections: ‘super-antibodies’ enter the fray. Nat Rev Immunol. 2018; 18(5): 297–308. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPREVAIL II Writing Group, Multi-National PREVAIL II Study Team, Davey RT Jr, et al.: A Randomized, Controlled Trial of ZMapp for Ebola Virus Infection. N Engl J Med. 2016; 375(15): 1448–1456. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou P, Yang XL, Wang XG, et al.: Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin. bioRxiv. 2020; 2020.01.22.914952. Publisher Full Text\n\nLi W, Moore MJ, Vasilieva N, et al.: Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature. 2003; 426(6965): 450–454. PubMed Abstract \| Publisher Full Text\n\nKarakus U, Pohl MO, Stertz S: Breaking the convention: Sialoglycan variants, Co-receptors and Alternative Receptors for Influenza A Virus Entry. J Virol. 2019; pii: JVI.01357-19. PubMed Abstract \| Publisher Full Text\n\nWong SK, Li W, Moore MJ, et al.: A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2. J Biol Chem. 2004; 279(5): 3197–3201. PubMed Abstract \| Publisher Full Text\n\nArbabi-Ghahroudi M: Camelid Single-Domain Antibodies: Historical Perspective and Future Outlook. Front Immunol. 2017; 8: 1589. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXia C, Gautam A: Biopharma CRO industry in China: landscape and opportunities. Drug Discov Today. 2015; 20(7): 794–798. PubMed Abstract \| Publisher Full Text\n\nDu L, Kou Z, Ma C, et al.: A truncated receptor-binding domain of MERS-CoV spike protein potently inhibits MERS-CoV infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines. PLoS One. 2013; 8(12): e81587. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang X, Mathieu M, Brezski RJ: IgG Fc engineering to modulate antibody effector functions. Protein Cell. 2018; 9(1): 63–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSondermann P, Szymkowski DE: Harnessing Fc receptor biology in the design of therapeutic antibodies. Curr Opin Immunol. 2016; 40: 78–87. PubMed Abstract \| Publisher Full Text\n\nDesmyter A, Farenc C, Mahony J, et al.: Viral infection modulation and neutralization by camelid nanobodies. Proc Natl Acad Sci U S A. 2013; 110(15): E1371–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoch K, Kalusche S, Torres JL, et al.: Selection of nanobodies with broad neutralizing potential against primary HIV-1 strains using soluble subtype C gp140 envelope trimers. Sci Rep. 2017; 7(1): 8390–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHe Y, Zhou Y, Liu S, et al.: Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine. Biochem Biophys Res Commun. 2004; 324(2): 773–781. PubMed Abstract \| Publisher Full Text\n\nGu J, Korteweg C: Pathology and pathogenesis of severe acute respiratory syndrome. Am J Pathol. 2007; 170(4): 1136–1147. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuba K, Imai Y, Rao S, et al.: A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury. Nat Med. 2005; 11(8): 875–879. PubMed Abstract \| Publisher Full Text\n\nSui J, Li W, Murakami A, et al.: Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association. Proc Natl Acad Sci U S A. 2004; 101(8): 2536–2541. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoore MJ, Dorfman T, Li W, et al.: Retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting enzyme 2. J Virol. 2004; 78(19): 10628–10635. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nImai Y, Kuba K, Rao S, et al.: Angiotensin-converting enzyme 2 protects from severe acute lung failure. Nature. 2005; 436(7047): 112–116. PubMed Abstract \| Publisher Full Text\n\nGu H, Xie Z, Li T, et al.: Angiotensin-converting enzyme 2 inhibits lung injury induced by respiratory syncytial virus. Sci Rep. 2016; 6: 19840. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZou Z, Yan Y, Shu Y, et al.: Angiotensin-converting enzyme 2 protects from lethal avian influenza A H5N1 infections. Nat Commun. 2014; 5: 3594. PubMed Abstract \| Publisher Full Text\n\nHaschke M, Schuster M, Poglitsch M, et al.: Pharmacokinetics and pharmacodynamics of recombinant human angiotensin-converting enzyme 2 in healthy human subjects. Clin Pharmacokinet. 2013; 52(9): 783–792. PubMed Abstract \| Publisher Full Text\n\nKhan A, Benthin C, Zeno B, et al.: A pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome. Crit Care. 2017; 21(1): 234. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi F, Li W, Farzan M, et al.: Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science. 2005; 309(5742): 1864–1868. PubMed Abstract \| Publisher Full Text\n\nLiu P, Wysocki J, Souma T, et al.: Novel ACE2-Fc chimeric fusion provides long-lasting hypertension control and organ protection in mouse models of systemic renin angiotensin system activation. Kidney Int. 2018; 94(1): 114–125. PubMed Abstract \| Publisher Full Text\n\nYasui F, Kohara M, Kitabatake M, et al.: Phagocytic cells contribute to the antibody-mediated elimination of pulmonary-infected SARS coronavirus. Virology. 2014; 454–455: 157–168. PubMed Abstract \| Publisher Full Text\n\nGlowacka I, Bertram S, Herzog P, et al.: Differential downregulation of ACE2 by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus NL63. J Virol. 2010; 84(2): 1198–1205. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi W, Zhang C, Sui J, et al.: Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2. EMBO J. 2005; 24(8): 1634–1643. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChamow SM, Duliege AM, Ammann A, et al.: CD4 immunoadhesins in anti-HIV therapy: new developments. Int J Cancer Suppl. 1992; 7: 69–72. PubMed Abstract\n\nShearer WT, Israel RJ, Starr S, et al.: Recombinant CD4-IgG2 in human immunodeficiency virus type 1-infected children: phase 1/2 study. The Pediatric AIDS Clinical Trials Group Protocol 351 Study Team. J Infect Dis. 2000; 182(6): 1774–1779. PubMed Abstract \| Publisher Full Text\n\nJacobson JM, Lowy I, Fletcher CV, et al.: Single-dose safety, pharmacology, and antiviral activity of the human immunodeficiency virus (HIV) type 1 entry inhibitor PRO 542 in HIV-infected adults. J Infect Dis. 2000; 182(1): 326–329. PubMed Abstract \| Publisher Full Text\n\nGardner MR, Kattenhorn LM, Kondur HR, et al.: AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges. Nature. 2015; 519(7541): 87–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGardner MR, Fellinger CH, Kattenhorn LM, et al.: AAV-delivered eCD4-Ig protects rhesus macaques from high-dose SIVmac239 challenges. Sci Transl Med. 2019; 11(502): pii: eaau5409. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShao Z, Shrestha K, Borowski AG, et al.: Increasing serum soluble angiotensin-converting enzyme 2 activity after intensive medical therapy is associated with better prognosis in acute decompensated heart failure. J Card Fail. 2013; 19(9): 605–610. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWan Y, Shang J, Sun S, et al.: Molecular mechanism for antibody-dependent enhancement of coronavirus entry. J Virol. 2019; pii: JVI.02015-19. PubMed Abstract \| Publisher Full Text\n\nRaj VS, Mou H, Smits SL, et al.: Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature. 2013; 495(7440): 251–254. PubMed Abstract \| Publisher Full Text" }	[ { "id": "59692", "date": "11 Feb 2020", "name": "Yi-Wei Tang", "expertise": [ "Reviewer Expertise Molecular and diagnostic virology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis review provide an authoritative review on 2019-nCoV infection therapeutic options. After briefly covered several treatment strategies, the author focused on options to block virus from entering cells. Figure 1 is terrific.\nI suggest that the authors provide a table contrasting the four strategies mentioned at the beginning.\n\nIn the treatment involving neutralizing antibodies, the author should discuss potential side effect such as antibody dependent enhancement (ADE).\n\nSo many therapeutic options have been mentioned in China including Chinese herbs and chlorquine. It will be great if the author can briefly review and comment on this.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "59434", "date": "18 Feb 2020", "name": "Huihui Mou", "expertise": [ "Reviewer Expertise Viral entry", "coronavirus virus receptors and S protein", "use of receptors as entry inhibitors" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA very sensible opinion article based on information well known to the coronavirus field but perhaps not well appreciated by others who might want to address this potential pandemic.\n\nI found no factual or interpretive errors. Were the issue not so pressing, I might have somewhat discounted the article based on 'obviousness' but this is obviousness to the field and not to the wider group of readers.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	2	https://f1000research.com/articles/9-72
https://f1000research.com/articles/9-87/v1	06 Feb 20	{ "type": "Opinion Article", "title": "Upholding confidentiality in the preparation and distribution of medication in prisons: implementing recommendations of the European Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment", "authors": [ "Nguyen Toan Tran", "Hans Wolff", "Hans Wolff" ], "abstract": "Confidentiality must be ensured even in the preparation and distribution of medications in detention settings. In this respect, the European Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment found during recent prison visits several instances where prison staff, and at times detainees, dispensed prescribed treatments and supervised their intake. Such a practice compromises medical confidentiality requirements and the establishment of a trusting doctor-patient relationship. To respect medical confidentiality and ensure safety and quality of care, the authors argue that only qualified healthcare personnel should prepare and distribute prescribed medications, all of which require specialized training. They call for robust research that examines the operational barriers and facilitators as well as the respect of human rights related to various approaches to medication preparation, distribution, and intake so that people in detention can access their treatment with safety, confidentiality, autonomy, and dignity.", "keywords": [ "Access to medication", "preparation", "distribution", "detention", "prison", "confidentiality", "autonomy", "human rights", "CPT", "equivalence of care", "professional independence." ], "content": "Background\n\nThe European Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment (CPT) provides a non-judicial preventive mechanism to protect persons deprived of liberty against torture and other forms of ill-treatment1. The CPT visits detention places in the 47 member states of the Council of Europe. The visits include the assessment of medical services in carceral settings to gain insights into the way individuals access healthcare prevention, treatment, and care. The access to healthcare services for individuals experiencing incarceration is essential as this priority population carries a high burden of physical and mental health conditions2–5. Operating such services in prison must be underpinned by the following guiding principles: timely access to competent medical staff who work independently from the criminal justice system6, equivalence of preventive and curative care services that are free-of-charge to patients (i.e., states bear all the healthcare costs), humanitarian assistance to individuals with increased vulnerabilities (e.g., mothers with children, adolescents), and patient’s right to informed consent and confidentiality7. Failing to respect these principles can result in situations falling within the scope of inhumane and degrading treatment. The objective of this article is to underscore the paramountcy of upholding confidentiality in the preparation and distribution of medication, as highlighted by recent CPT visits.\n\n\nExamples of violations\n\nThere is a paucity of published peer-reviewed literature on this subject and our sources stem from published CPT reports. Once prescribed, medications require coordinated preparation and distribution for individuals to have timely access to their treatment. In Scotland, the CPT delegation found that medical and prison staff shared the task of distributing medication and supervising its intake8. In Greece, prison staff and detained individuals were acting as orderlies (i.e., they were trained in first aid and selected clinical tasks), who dispensed medication under the supervision of nurses and had access to patient medical records9. In Norway, it was the duty of the custodial officers to distribute prescribed medications despite the daily presence of a nurse10. In Estonia, nurses prepared the medication, and custodial staff ensured its distribution except for psychotropic treatment, which was delivered by nurses11. In Cyprus, several prison officers worked as medical orderlies who distributed medication and accompanied doctors on their rounds, with a few assigned to medical duty during the day or at night12. In the Netherlands, external pharmacies prepared the medication and custodial staff carried out its distribution. For prescribed opioids and psychiatric medication, prison officers also had to check that recipients swallowed them properly (patients reported that they had occasionally received the wrong medication, for example that of their neighbor, due to a lack of attention by the prison officer)13.\n\n\nCPT rules and recommendations\n\nCooperation between healthcare professionals and prison authorities is necessary if it is deemed reasonable and respects the guiding principles mentioned above. However, in the given examples, the distribution of medication, including psychotropic substances and methadone, by prison officers or by incarcerated persons compromised the respect of medical confidentiality—the medication name and its dosage were visible. Such a practice could compromise medical confidentiality requirements and the establishment of a proper doctor-patient relationship.\n\nAccording to the 1992 CPT report, there should be appropriate supervision of the pharmacy and the distribution of medicines. Further, the preparation of medicines should always be entrusted to qualified staff (pharmacist/nurse, etc.)7 In other words, to respect medical confidentiality and ensure safety and quality of care, individuals in prison and custodial staff should not be involved in performing healthcare tasks and only qualified healthcare personnel should prepare and distribute prescribed medications, all of which require specialized training.\n\nWe acknowledge the difficulty in following this recommendation, especially in smaller detention facilities, where healthcare professionals are not available 24h/7 when compared to larger and high security facilities. These facilities tend to have the most comprehensive healthcare services and staffing because the patient population is older and present for a longer period14. However, irrespective of the size of the detention centers, people experiencing incarceration use health services more often when compared with the community15. We also understand that ensuring access to medication while respecting confidentiality, complying with prison security requirements, and accounting for fears of theft, trafficking, and misuse, especially of psychoactive medication16, needs a balanced operational approach.\n\n\nRecommendations for best practice\n\nThe best-case scenario, one that respects medical confidentiality and quality of care while empowering patients in their autonomy, would comprise the following steps: as blister medication increases adherence to treatment17, prescribed medications are left in their blisters (for tablets) and packaged individually for each patient by healthcare staff (such as in the detention facilities of Geneva, Switzerland) or an automated pharmacy preparation system (such as in larger facilities in France); to avoid the confusion of roles between healthcare and prison staff18 and to respect patients’ right to confidentiality19, healthcare professionals—not prison officers or incarcerated individuals—distribute the medications either in-hand (as privately and confidentially as possible) or in individual lockable medication boxes that each user can independently access (such as at La Brenaz facility in Geneva); and when supervised intake is not medically indicated, patients should have the option of taking their treatment in the privacy of their cells.\n\nHowever, based on the CPT experience, there are limited examples that adhere to this best-case scenario. Therefore, we call for robust operational research that examines the operational barriers and facilitators as well as the respect of human rights related to various approaches to medication preparation, distribution, and intake. Identifying and disseminating best practices that are rights-based will allow people in detention to access their treatment with safety, confidentiality, autonomy, and dignity.\n\n\nData availability\n\nNo data are associated with this article.\n\n\nDisclaimer\n\nAlthough HW is a member of the CPT, the opinions expressed in this article reflect those of the authors and are based on CPT reports and recommendations.", "appendix": "References\n\nEuropean Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment: About the CPT. [Accessed on 24 October 2019]. Reference Source\n\nFazel S, Baillargeon J: The health of prisoners. Lancet. 2011; 377(9769): 956–65. PubMed Abstract \| Publisher Full Text\n\nDudeck M, Drenkhahn K, Spitzer C, et al.: Traumatization and mental distress in long-term prisoners in Europe. Punishm Soc. 2011; 13(4): 403–23. Publisher Full Text\n\nFazel S, Bains P, Doll H: Substance abuse and dependence in prisoners: a systematic review. Addiction. 2006; 101(2): 181–91. PubMed Abstract \| Publisher Full Text\n\nFazel S, Seewald K: Severe mental illness in 33,588 prisoners worldwide: systematic review and meta-regression analysis. Br J Psychiatry. 2012; 200(5): 364–73. PubMed Abstract \| Publisher Full Text\n\nPont J, Enggist S, Stöver H, et al.: Prison Health Care Governance: Guaranteeing Clinical Independence. Am J Public Health. 2018; 108(4): 472–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEuropean Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment: Health care services in prisons - 3rd General Report of the CPT. Council of Europe, 1993. Reference Source\n\nEuropean Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment: Report to the Government of the United Kingdom on the visit to the United Kingdom carried out by the European Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment (CPT) from 17 to 25 October 2018; Para 107. Strasbourg: CPT. 2019.\n\nEuropean Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment: Report to the Greek Government on the visit to Greece carried out by the European Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment (CPT) from 14 to 23 April 2015; Para 80, 81, 85. Strasbourg: CPT. 2016. Reference Source\n\nEuropean Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment: Report to the Norwegian Government on the visit to Norway carried out by the European Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment (CPT) from 28 May to 5 June 2018; Para 90. Strasbourg: CPT. 2019. Reference Source\n\nEuropean Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment: Report to the Estonian Government on the visit to Estonia carried out by the European Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment (CPT) from 27 September to 5 October 2017; Para 60. Strasbourg: CPT. 2019. Reference Source\n\nEuropean Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment: Report to the Government of Cyprus on the visit to Cyprus carried out by the European Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment (CPT) from 2 to 9 February 2017; Para 85-86. Strasbourg: CPT. 2018. Reference Source\n\nEuropean Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment: Report to the Government of the Netherlands on the visit to the Netherlands carried out by the European Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment (CPT) from 2 to 13 May 2016; Para 60. Strasbourg: CPT. 2017. Reference Source\n\nMarshall T, Simpson S, Stevens A: Health care in prisons: a health care needs assessment. University of Birmingham Birmingham; 2000. Reference Source\n\nMarshall T, Simpson S, Stevens A: Use of health services by prison inmates: comparisons with the community. J Epidemiol Community Health. 2001; 55(5): 364–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWhite N, Ali R, Larance B, et al.: The extramedical use and diversion of opioid substitution medications and other medications in prison settings in Australia following the introduction of buprenorphine-naloxone film. Drug Alcohol Rev. 2016; 35(1): 76–82. PubMed Abstract \| Publisher Full Text\n\nConn VS, Ruppar TM, Chan KC, et al.: Packaging interventions to increase medication adherence: systematic review and meta-analysis. Curr Med Res Opin. 2015; 31(1): 145–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPont J, Stöver H, Wolff H: Dual loyalty in prison health care. Am J Public Health. 2012; 102(3): 475–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElger BS, Shaw DM: Confidentiality in prison health care–A practical guide. Emerging Issues in Prison Health. Springer. 2017; 183–200. Publisher Full Text" }	[ { "id": "62164", "date": "17 Apr 2020", "name": "Nickolas Zaller", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, this is a very well written commentary with some important implications. I do think there could be a few additional considerations incorporated in order to broaden the applicability of the perspective and recommendations. For example, in some parts of the world (e.g. US) correctional facilities contract medical services to a third-party vendor. As such, there needs to be additional coordination between corrections-based staff and non-corrections based medical staff, especially with respect to facilitating medical appointments and medication receipt. With respect to the recommendations for best practice, while the best-case scenario the authors outline is one that all facilities should strive to achieve, it would be nice to see some gradation of compliance with this best-practice in order to make it a bit more practical and/or feasible. For example, if facilities do not have the resources, especially in terms of medical staff to adequately supervise medication, what can be done to mitigate concerns re breaches in confidentiality? It would be good to see recommendations for facilities across a “spectrum” of compliance such that those facilities in poor resourced settings do not feel as though they must undertake an all or nothing approach. Again, if facilities can take measured steps toward achieving the stated best-case scenario, they should be strongly encouraged to do so.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "174975", "date": "01 Nov 2023", "name": "Joerg Pont", "expertise": [ "Reviewer Expertise Prison health care", "medical ethics in prison health care", "Internal medicine" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article is a concise presentation of a much debated issue in many penitentiary health care services. Due to shortage of health care professionals several penitentiary systems resort to non-medical staff or even inmates for dispensing prescibed medication, a practice that clearly is in conflict with the professionally indispensible ethical principle of medical confidentiality and that violates the patients' right to have their medical data kept in medical confidentiality.The article also provides practical recommendations how to overcome this problem.\nFor the same reason, the authors may consider also recommending for medically indicated supervised intake of medication that the supervison should be conducted by health care staff and not by custodial staff and, if presence of custodial staff is deemed necessary, they should stay outside of the room.\nPlease correct in the text the year of the 3rd General Report of the CPT in accordance to ref 7, i.e., 1993 not 1992.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-87
https://f1000research.com/articles/8-1435/v1	14 Aug 19	{ "type": "Research Article", "title": "Diversity and distribution of fish fauna of upstream and downstream areas at Koto Panjang Reservoir, Riau Province, Indonesia", "authors": [ "Netti Aryani", "Indra Suharman", "Azrita Azrita", "Hafrijal Syandri", "Ainul Mardiah", "Indra Suharman", "Azrita Azrita", "Hafrijal Syandri", "Ainul Mardiah" ], "abstract": "Background: The capture fishery sectors in the river and reservoir play an important role in the Indonesian economy through increased income and diversification of livelihoods. The present study was conducted to ascertain fish diversity and their distribution pattern in the upstream and downstream areas of Koto Panjang Reservoir, Riau Province-Indonesia. Methods: Fish samples were collected for a period of 12 months using a variety of fish nets at four sites; Koto Mesjid (KM) and Batu Bersurat (BB), located in the upstream area of Koto Panjang Reservoir and Rantau Berangin (RB) and Kuok (KK), located in the downstream area of Koto Panjang Reservoir. Data obtained were analyzed using standard taxonomic keys based on morphometric characters. Results: A total of 44 species belonging 19 families and 33 genera were recorded in the study area. Alpha diversity indices showed that fish diversity in this area was quite high (Shannon’s index = 2.10 and Simpson-D = 0.21) and evenness was low (evenness H/S =0.19). The ﬁsh in KM and BB sites (upstream) were from eight and 11 families, respectively. In RB and KK sites (downstream), fish were from 16 and 15 families, respectively. In KM, BB, RB and KK sites, the dominant family was Cyprinidae, comprising 33.45%, 50.95%, 43.04% and 39.35% of all fish caught at each site, respectively. Exotic species, especially Nile tilapia, were 20.15%, 14.11%, 5.62%, and 5.34%, respectively. Some differences were also noted between the upstream and downstream reservoirs, with a slight increase in exotic species in the upstream reservoir over the study period (from 11.39% vs. 34.66%), corresponding to decrease of native species (from 88.61% vs. 65.34%). Conclusions: The diversity and distribution of fish fauna were varied in upstream and downstream areas of Koto Panjang Reservoir. The exotic species were found to be dominant in the upstream reservoir areas.", "keywords": [ "Ichthyodiversity", "shannon’s index", "evenness", "exotic", "reservoir", "river" ], "content": "Introduction\n\nThe capture fishery and aquaculture sectors play an important role in the Indonesian economy through increased income, diversification of livelihoods, supply of animal protein, and foreign exchange earnings. In 2015, the total fishery production of Indonesia was 16,954,344 metric tons, of which 455,270 metric tons was obtained from inland capture fisheries, 6,065,060 metric tons was obtained from marine fisheries, and 10,074,014 metric tons was obtained from aquaculture fisheries production1. A total of 1,300 fish, including 40 endemic species are known to inhabit in the freshwaters of Indonesia, with 16 exotic species recorded in Indonesia2. The production from the inland capture fisheries of Indonesia comes from wetlands (rivers, lakes, swamps, oxbow lakes, floodplains, etc). In Riau Province, one of the rivers that produce freshwater fish from capture fishery is the Kampar Kanan river. At present, major Cyprinidae species such as Leptobarbus hoevenii, Osteochilus haselti and Rasbora argyrotaenia, along with exotic species, such as common carp (Cyprinus carpio) and Nile tilapia (Oreochromis niloticus), are the available species of fish at local food markets.\n\nAccording to Mulyadi3, Kampar Kanan river is one of the largest rivers in Riau Province. It is approximately 213.5 km long and between 125 to 143 m wide, with significant capture fishery potential. Since 1996, this river has been dammed into a reservoir (Koto Panjang Reservoir) for the operation of a 114 MW hydroelectric power plant. The dam height is 96 m and located at altitude of 85 m above sea level3, and at the geographical position 0o17'23.76˝N and 100o52'53.39˝E. However, at the location of the dam, there is no fishway. The abiotic and biotic characteristics of river ecosystems can be affected by the construction of dams. These conditions have an impact on mortality and failure of fish migration4–7. The hydrologic regime of streams changing from lotic to lentic can influence the water retention in the reservoir. In general, the lentic condition causes a decrease in native species and then an increase in exotic species8,9. Furthermore, the degradation of aquatic fauna habitats can be caused by an increase in homogeneity of water channels, which has an effect on the seasonal flow variability of river10. The reduction in river runoff also affects the habitat and distribution of fish fauna.\n\nOn the other hand, there are serious threats to the original fish biodiversity in the downstream and upstream regions of the reservoir due to the dam of the hydroelectric power plant, such as sand mining in river, land use change and aquaculture activity with cages, which can affect the depth of river water, food availability, and breeding sites. Amadi et al.11 state that original biodiversity can be eroded by habitat degradation and alien species impact. Meanwhile, aquaculture heavily impacts the structure and diversity of local fish communities12,13. Hence, it was essential to study fish diversity continuously in different ecosystem areas, including upstream and downstream areas at Koto Panjang Reservoir, Riau Province, Indonesia.\n\n\nMethods\n\nThere are no required permits from the government of the Republic of Indonesia to capture the species in this study in the upstream and downstream regions of Koto Panjang Reservoir. The study was funded by LPPM (Research and Community Service) University of Riau under Directorate of Research and Community Service, Ministry of Research Technology and Higher Education Republic of Indonesia with contract no. 767/UN.19.5.1.3/PT.01.03/2018. This grant included ethical approval and permits to collect fish samples including native species (endangered and non-endangered) and exotic fish species. Specimens of fish species categorized as non-endangered and exotic were killed once caught. Endangered fish species (Hemibagrus wyckii) were returned to the river in good condition following analysis in the field. All efforts were made to ameliorate any animal suffering through anaesthetizing fish with ice water before euthanization.\n\nFish sampling was carried out from January to December 2018 at four sites, namely, Koto Mesjid (KM) and Batu Bersurat (BB) (upstream reservoir), Rantau Berangin (RB) and Kuok (KK) (downstream reservoir) (Table 1). Fish samples were randomly collected from the study area using traditional fishing gear (e.g. traps nets, cast nets, gill nets, drag nets, and fishing poles). Data was collected once a month at each site and five pieces of fishing gear were in operation at any one time.\n\nTrap nets (local name bubu) are made from bamboo woven with rattan and have a cylindrical front with a diameter of 80 cm and cone-shaped back, with a length of two meters. Chicken intestine was placed inside the gear as bait. This gear was used between the hours of 18:00 and 06:00 at the bottom of river and reservoir to catch demersal fish such as Bagridae, Pangasidae, Gobitidae, Claridae, Anabantidae, Belontiidae.\n\nCast nets (local name Jala) are a type of active fishing gear made from string, with a length of 2.5 meters and mesh size of 1.5 inches. This gear was used on the river and reservoir sides by fishermen using canoes. This gear was operated during the day from 06.00 until 10.00. The purpose of cast nets is to catch the family of Cyprinidae, Osphronemidae, Notopteridae and Cichlidae.\n\nGill nets (local name jaring insang) are made of rectangular monofilament yarn, are 60 meters in length and 8 meters in depth, with a 2.5 inch mesh size. These were operated passively and transversely on the surface of river from 18.00 until 06.00 to catch pelagic fish such as Cyprinidae, Osphronemidae, Notopteridae and Cichlidae.\n\nA drag net (local name belad) is a passive fishing device made from nylon net material with a diameter of 0.15 mm and a mesh size of 0.5 inches. This gear is assisted by bamboo or wood as a cantilever, with a height of 2.5 meters and a length of 100 meters, which was placed parallel to the river coastline from 18.00 until 06.00. The purpose of the drag net is to catch the family of Bagridae, Pangasidae, Gobitidae, Claridae, Anabantidae, Belontiidae and Siluridae.\n\nThe fishing pole (local name rawai) used consisted of a main line with a length of 50 to 100 cm and a distance from one branch line to another of 1.5 meters. One fishing pole has hooks ranging from 20 to 30 pieces and the hooks are size no. 15. The fishing pole was operated passively on the river bottom between the hours of 18.00 and 06.00 and used chicken intestine as bait. The main purpose of fishing pole is to catch the family of Bagridae, Tetradontidae, Pangasidae and Channidae.\n\nSamples were classified as endangered, non-endangered and exotic fish species based on the categories described by Kottelat and Whitten14. Once caught, fish were anaesthetized in ice water at a temperature of 5°C. Euthanization was achieved by piercing part of the brain of the fish. Samples were given an intra-peritoneal injection prior to store in a formalin solution. Smaller specimens were stored directly in 5% formalin solution, while the larger specimens were stored in 10% formalin solution. Specimens that were categorized as non-endangered were transported in a cold box (10 °C) to the Fish Biology and Ichthyology Laboratory, Department of the Aquaculture, Riau University for measurement of specimen length, weight, and morphometric characteristics. Endangered fish species such as Hemibagrus wyckii were analyzed and measured in the field. Then, the same fish was returned to the river in good condition. The length, body weight and morphometric characteristics were only collected for 10 individual specimens from each species.\n\nSamples from each site were separately packed in labeled plastics jars according to date, site, time, and locality. Each specimen was labeled with a specific number manually. Classification and taxonomic identification of the sampled specimens was completed using standard keys15,16 on the basis of morphometric and meristic characters.\n\nThe data of different species for abundance and occurrence was calculated for species richness (S), Shannon diversity Index (H’), Simpson diversity index (D), evenness (H/S) and Sorenson’s coefficient (CC)17–19 using Microsoft Excel 2010 (version 14.0). The accuracy of the data and results were verified by applying all the diversity indices separately according to sampling months and sampling sites.\n\n\nResults\n\nDuring the study, forty-four different species of fish were collected from the study area. A total of 8017 specimens of fish were collected from four sites20. The details of the fish species collected on a monthly basis for the period of one year (January to December 2018) are presented in Table 2. The highest number of fish collected during one month was collected during August 2018 (873 specimens), followed by the months of September > July > June > October > May > November > April > March > December > February > January.\n\nA total of 44 species belonging to 19 families and 33 genera were sampled from the four sites over one year in the upstream and downstream areas at Koto Panjang Reservoir. There were seventeen species which are commercially important, as determined by their high market value. These species including ornamental fish species such as Chromobotia macrachantus, Chromobotia hymenophysa, Thrichogaster trichopterus and Mystus micracanthus. The highest ichthyodiversity in study area was calculated during July and August 2018 (44 species), followed by June and September (42 species), May and October (41 species), April and November (39 species), December (38 species), March (36 species), February (30 species) and January (26 species). Numerically, the most abundant and diverse family was Cyprinidae, comprising of 16 species, followed by Bagridae and Channidae (represented by four species each). The fourth most diverse family was Gobitidae, represented by three species in the study area. The least diverse families were Claridae, Pangasidae, Anabantidae, Mastacembelidae, Osphronemidae, Pristolepididae, Cygnolossidae, Notopteridae, Hemiramphidae and Cichlidae, represented by only one species for each (Table 2). Barbodes schwanifeldi, Hemibagrus nemerus, Ompok hypophthalmus, Rasbora argyrotaenia and Oreochromis niloticus were recorded as the most abundant species, comprising 5.88%, 6.20%, 6.71%, 8.76% and 9.80% of all fish caught, respectively. The least abundant were Channa pleurothalmus, Hemibagrus wyckii and Pangasius pangasius, representing 0.06%, 0.08% and 0.09%, respectively.\n\nTable 3 shows different diversity indices used to calculate the species abundance data. The highest species richness was recorded between the months of June and September. Similarly, the highest values for Shannon’s diversity index (H’) were achieved during October 2018 (2.27), August and November 2018 (2.23), and the lowest values during January and February 2018 (1.74 and 1.91).\n\nThe highest Simpson diversity index (I/D) was recorded during the months of January (0.25), followed by April (0.23), February, March and May (0.22), and the lowest value was recorded in the months of October and November (0.18). Similarly, the highest values of species evenness (H/S) was recorded in the month of March > December and its least value was recorded during the month of June. Furthermore, the detailed values of different diversity indices on the basis of sampling sites were given in the Table 4. Whereas, the Sorenson’s coefficient (CC) between upstream and downstream areas at Koto Panjang Reservoir were presented in Table 5. The commercially important fish species captured in downstream Reservoir were Hemibagrus wyckii, Hemibagrus nemurus, Wallago leerii, Pangasius pangasius, Osphronemus gourami, Puntioplites bulu, Rasbora argyrotaenia, Channa striata, and Channa micropeltes. Whereas, in the upstream Reservoir found were Pristilepis grooti, Oxyeleotris marrmorata, Hemibagrus nemurus and Channa striata and Oreocromis niloticus.\n\nNote: KM = Koto Mesjid, BB = Batu Bersurat, RB = Rantau Berangin, KK = Kuok.\n\nNote: KM = Koto Mesjid, BB = Batu Bersurat, RB = Rantau Berangin, KK = Kuok.\n\nThe abundance and number of families found between sites was varied. At KM and BB (upstream reservoir), 8 and 11 families were found, respectively, while at RB and KK (downstream reservoir), 16 and 15 families were found, respectively (Figure 1). The dominant families in each site was Cyprinidae, comprising 33.45%, 50.95%, 43.04% and 39.35% of all fish caught at KM, BB, RB and KK, respectively. Whereas, the exotic species, especially Nile tilapia (O. niloticus), in each site comprised 14.11%, 20.15%, 5.62% and 5.34% of all fish caught at KM, BB, RB and KK, respectively. Some differences were also noted between the upstream and downstream reservoirs areas (Figure 2), with a slight increase of exotic species in the upstream reservoir (from 11.39% to 34.66%) and a corresponding decrease of native species (from 88.61% to 65.34%).\n\na) Koto Mesjid, b) Batu Bersurat, c) Rantau Berangin and d) Kuok.\n\n\nDiscussion\n\nOur results showed that the highest abundance and diversity of fish species collected in June, July and August, which may be due to a lesser water current in the study area during these dry season months. According to Kriaučiūnienė et al.21 the abundance of fish species in rivers can be affected by river discharge. However, future alterations in river water temperature will have a significantly larger influence on the abundance of fish than river discharge. Overexploitation and illegal sand mining have affected the abundance of fish in the Kampar Kanan river22. Furthermore, the fundamental measures of aquatic ecosystems, including species richness and diversity indices, are influenced by the alterations in abiotic factors such as river water temperature and discharge23–26.\n\nOur study also revealed significantly lower species diversity in KM and BB (upstream reservoir) compared to that of RB and KK (downstream reservoir). The overall richness in KM was much lower than that found in RB (18 vs. 37). During the research period, we also recorded a slight increase in exotic species in the upstream reservoir and a decrease in native species. In contrast, in a Portuguese reservoir, it was found that there was a slight increase in exotic species in the downstream reservoir4. This result might be a consequence of cumulative impacts of cultivation of fish in floating net cages, such as Nile tilapia and common carp. According to Russel et al.27, Nile tilapia cause the extinction of native fish species by preying on eggs, fry and small fish of other species. On the other hand, the decrease in species richness can be influenced by aquaculture activity, such as water quality degradation, intensified competition, invasive species, and habitat fragmentation11,28–30. Koto Panjang Reservoir was categorized as eutrophic, with level index values ranging from 4.6-5.231. According to Edwards32, species with high environmental tolerance would be survive in poor environmental conditions, such as high pollution caused by food waste at the reservoir, while the sensitive species will disappear. Furthermore, fish populations in a reservoir can be affected by hydropower dams3,4. In addition, the intensifying competition for food and space between wild species and the large number of cultured species will lead to a decrease in wild fish numbers33,34.\n\nOur study confirmed the existence of three species, Pangasius pangasius, Wallago leerii and Chitala hypselonotus, at Koto Panjang Reservoir that were not found previously by Warsa et al.35 and Krismono et al.36. The study area also represents the area of the Kampar Kanan river with the largest fish species, including W. leerii, H. wyckii and P. pangasius. The construction of the dam for power generation purposes is posing serious threats to the biodiversity of Kampar Kanan river. After the construction of the Koto Panjang dam on Kampar Kanan river, the movement of fish upstream has been restricted. Osteochilus kelabau has not been reported at RB in our study although this species has previously been found in this area37. Similarly, the population of H. wyckii has also been dramatically reduced and restricted between Koto Panjang Reservoir barrages and Kampar Kanan river after the construction of these barrages. H. wyckii in the Kampar Kanan river is categorized as ‘vulnerable to endangered’38. According to Piria et al.39, the disturbances to the fish assemblage pattern have coincided with the presence of multiple stressors of human origin, such as pollution, flood protection and dam construction. Meanwhile, dams can of change the hydrological dynamics, patterns of biological production, loss of native species in the downstream regions and distribution of organisms in space and time3,5,10,40,41.\n\nThere are a number of inadvertently introduced fish species in the upstream and downstream Reservoir, such as O. niloticus, Cyprinus carpio, Leptobarbus hoevenii and Pangasius hypophthalmus, while rest of the 40 species belong to the native fish fauna of the Kampar Kanan river37. The unique feature of the abundance data was that the exotic family Cichlidae is well established and its population is increasing day by day, especially in the upstream areas at Koto Panjang Reservoir. In recent years, the Koto Panjang Reservoir has had very important roles, such as housing power plants with a capacity of 114 MW and serving as a fishery capture and aquaculture area with floating net cage farming, especially for the cultivation of O. niloticus. Cichlidae was the third most abundant family. These alien fish species represent a significant risk for the local fish community and other aquatic animals. Gu et al.30 state that the invasion of O. niloticus negatively affected the fishery economy and native fish species in the Pearl River of Guangdong Province, China.\n\nIn addition to the species richness (S) analysis, Shannon diversity index (H’), Simpson diversity index (D) and evenness (H/S), we also analyzed the Sorenson’s coefficient (CC) between the upstream and downstream sites. The Sorenson’s coefficient for each site varied between 0.54 and 0.92. The highest value of Sorenson’s coefficient was recorded between KM and RB, at 0.92, and the lowest value was recorded between BB and RB, at 0.54 (Table 5). According to Sorenson’s coefficient, these communities have quite a bit of overlap or similarity.\n\nThe high diversity of native species in the water body will decrease their tolerance in poor aquatic environments. In contrast, the invasive fish species have a high tolerance to poor water quality42. The interaction between different species, combined with the limnological and physical properties of the aquatic ecosystem, may influence the diversity and distribution of fish fauna43. In this study, the diversity of fish to be found smaller than Fhitra and Siregar44, who reported 58 fish species belonging to 23 families and 40 genera from Kampar Kanan river. Simanjuntak et al.45 described 86 species and 21 families in the Kampar Kiri river of the Kampar District. On the other hand, Nurdawati et al.46 reported 96 species in the Batanghari river, Indonesia. Additionally, Bahri47 described 86 species in the Musi river and Kottelat and Whitten48 reported 1300 species of freshwater fish across Indonesia that live in wetlands (rivers, lakes, bogs, oxbow lakes, floodplains, etc.).\n\n\nConclusion\n\nThe results indicate that the diversity and distribution of fish fauna in upstream and downstream areas of the Koto Panjang Reservoir were varied, and the evenness was low. The abundance and composition of fish in each site was dominated by Cyprinidae families, although exotic species were more dominant in the upstream reservoir compared to the downstream reservoir areas. Therefore, the management of the river and reservoir in a more holistic manner is important, for example, the management of land use, sand mining and aquaculture activity, as well as possible habitat restorations. All the factors above are a prerequisite for the environmental sustainability and conservation of fish diversity in the upstream and downstream areas at Koto Panjang reservoir and other regions.\n\n\nData availability\n\nFigshare: Row data fish fauna at upstream and downstream. https://doi.org/10.6084/m9.figshare.8964284.v120\n\nThis project contains the following underlying data:\n\n– Tables 2 – 5 (raw data for ichthyodiversity of fish in each site).\n\n– Table 6 (data for abundance and composition of aquatic fauna each sites in the upstream and downstream areas at Koto Panjang Reservoir)\n\n– Table 7 (data for grand total and percentage of families of aquatic fauna in the upstream areas at Koto Panjang Reservoir)\n\n– Table 8 (data for grand total and percentage of families of aquatic fauna in the downstream areas at Koto Panjang Reservoir)\n\n– Table 9 (character morphometric and meristic of fishes of upstream and downstream areas at Koto Panjang Reservoir, Riau Province-Indonesia)\n\n– Table 10 (sample sizes of fish populations (n=10) in the upstream and downstream areas at Koto Panjang Reservoir on January to December 2018)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThis study was funded by a study grant (Riset Dasar Unggulan Perguruan Tinggi) from the Directorate of Research and Community Service, Ministry of Research Technology and Higher Education Republic of Indonesia [767/UN.19.5.1.3/PT.01.03/2018].\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors thank the Ministry of Research Technology and Higher Education Republic of Indonesia for supporting this study through the competitive grants scheme (Riset Dasar Unggulan Perguruan Tinggi) 2019. The appreciation goes to all of fisherman and students who helped the author during data collection in field.\n\n\nReferences\n\nCSI: Center for data, statistics and information (CSI). Marine and Fisheries in Figures. Ministry of Marine and Fisheries Republic of Indonesia. 2015. Reference Source\n\nFFS: Fish Quarantine Agency, Fisheries Product Quality and Safety Control (FFS). Ministry of Marine and Fisheries Republic of Indonesia. 2015.\n\nMulyadi A: Electrical industry of Hydroelectric power plant Koto Panjang VS environmental problems. Jurnal Industri dan Perkotaan. 2003; 13(8): 625–631. Reference Source\n\nSantos RMB, Sanches Fernandes LF, Cortes RMV, et al.: Integrative assessment of river damming impacts on aquatic fauna in a Portuguese reservoir. Sci Total Environ. 2017; 601–602, 1108–1118. PubMed Abstract \| Publisher Full Text\n\nEloranta AP, Finstad AG, Helland IP, et al.: Hydropower impacts on reservoir fish populations are modified by environmental variation. Sci Total Environ. 2018; 618: 313–322. PubMed Abstract \| Publisher Full Text\n\nMarques H, Dias JHP, Perbiche-Neve G, et al.: Importance of dam-free tributaries for conserving fish biodiversity in Neotropical reservoirs. Biol Conserv. 2018; 224: 347–354. Publisher Full Text\n\nNyqvist D, Nilsson PA, Alenäs I, et al.: Upstream and downstream passage of migrating adult Atlantic salmon: Remedial measures improve passage performance at a hydropower dam. Ecol Eng. 2017; 102: 331–343. Publisher Full Text\n\nLi J, Dong S, Peng M, et al.: Effects of damming on the biological integrity of fish assemblages in the middle Lancang-Mekong River basin. Ecol Indic. 2013; 34: 94–102. Publisher Full Text\n\nWu H, Chen J, Xu J, et al.: Effects of dam construction on biodiversity: A review. J Clean Prod. 2019; 221: 480–489. Publisher Full Text\n\nZhao QH, Liu SL, Deng L, et al.: Assessing the damming effects on runoff using a multiple linear regression model: A case study of the Manwan Dam on the Lancang River. The 18th Biennial Conference of International Society for Ecological Modelling. Procedia Environmental Sciences. 2012; 13: 1771–1780. Publisher Full Text\n\nAmadi N, Petrozzi F, Kani GC, et al.: Freshwater fishes of Lower Guinean forest streams: Aquaculture heavily impacts the structure and diversity of communities. Acta Oecol. 2019; 94: 66–102. Publisher Full Text\n\nJiang Z, Wang C, Zhou L, et al.: Impacts of pen culture on alpha and beta diversity of fish communities in a large floodplain lake along the Yangtze River. Fish Res. 2019; 210: 41–49. Publisher Full Text\n\nLiao Y, Shou L, Jiang Z, et al.: Effects of fish cage culture and suspended oyster culture on macrobenthic communities in Xiangshan Bay, a semi-enclosed subtropical bay in eastern China. Mar Pollut Bull. 2019; 142: 475–483. PubMed Abstract \| Publisher Full Text\n\nKottelat M, Whitten AJ, Kartikasari SN, et al.: Freshwater Fishes of Western Indonesia and Sulawesi. Periplus Editions (HK) Ltd., Hong Kong, 1993; 221. Reference Source\n\nWeber M, De Beaufort: The Fishes of the Indo-Austrialian Archipelago II. E.J Brill Ltd. Leiden. 1913; 2: 404. Reference Source\n\nWeber M, De Beaufort: The Fishes of the Indo-Austrialian Archipelago III. E.J Brill Ltd. Leiden. 1916; 2: 404. Reference Source\n\nKrebs CJ: Ecology: The Experimental Analysis of Distribution and Abundance. Third Edition. Harper and Row Publishers. New York 776 1989. Reference Source\n\nOdum EP: Fundamental of Ecology. WB Saunders Co Publishing. New York. 1996.\n\nMagurran AE: Ecological Diversity and Its Measurement. New Jersey: Pricenton University Press. 1988. Publisher Full Text\n\nAryani N, Suharman I, Azrita A, et al.: Row data fish fauna at upstream and downstream. 2019. http://www.doi.org/10.6084/m9.figshare.8964284.v1\n\nKriaučiūnienė J, Virbickas T, Šarauskienė D, et al.: Fish assemblages under climate change in Lithuanian rivers. Sci Total Environ. 2019; 661: 563–574. PubMed Abstract \| Publisher Full Text\n\nAryani N, Suharman I, Syandri H: Reproductive performance of asian catfish (Hemibagrus wyckii Bagridae), a candidate species for aquaculture [version 1; peer review: 2 approved]. F1000Res. 2018; 7: 683. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParker J, Cao Y, Sass GG, et al.: Large river fish functional diversity responses to improved water quality over a 28 year period. Ecol Indic. 2018; 88: 322–331. Publisher Full Text\n\nSmith BJ, Simpkins DG: Influence of river plumes on the distribution and composition of nearshore Lake Michigan fishes. J Great Lakes Res. 2018; 44(6): 1351–1361. Publisher Full Text\n\nCrane DP, Kapuscinski KL: Capture efficiency of a fine mesh seine in a large river: Implications for abundance, richness, and diversity analyses. Fish Res. 2018; 205: 149–157. Publisher Full Text\n\nMuhammad H, Iqbal Z, Saleemi S: Diversity and distribution of fish fauna of Indus River at Taunsa Barrage in Punjab, Pakistan. Pakistan J Zool. 2018; 49(1): 155–161. Reference Source\n\nRussel DJ, Thuesen PA, Thomson FE: A review of the biology, ecology, distribution and control of Mozambique tilapia, Oreochromis mossambicus (Peters 1852) (Pisces: Cichlidae) with particular emphasis on invasive Australian populations. Rev Fish Biol Fish. 2012; 22(3): 533–554. Publisher Full Text\n\nFang JY, Wang ZH, Zhao SQ, et al.: Zheng. Biodiversity changes in the lakes of the Central Yangtze. Front Ecol Environ. 2006; 4(7): 369–377. Publisher Full Text\n\nHaddad NM, Brudvig LA, Clobert J, et al.: Habitat fragmentation and its lasting impact on Earth’s ecosystems. Sci Adv. 2015; 1(2): e1500052. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGu DE, Ma GM, Zhu YJ, et al.: The impacts of invasive Nile tilapia (Oreochromis niloticus) on the fisheries in the main rivers of Guangdong Province, China. Biochem Syst Ecol. 2015; 59: 1–7. Publisher Full Text\n\nHasibuan IF, Hariyadi S, Adiwilaga EM: Water Quality State and Trophic of PLTA Koto Panjang Reservoir, Riau Province. Jurnal Ilmu Pertanian Indonesia. 2017; 22(3): 147–155. Publisher Full Text\n\nEdwards P: Aquaculture environment interactions: past, present and likely future trends. Aquaculture. 2015; 447: 2–14. Publisher Full Text\n\nDing CZ, Jiang XM, Xie ZC, et al.: Seventy-five years of biodiversity decline of fish assemblages in Chinese isolated plateau lakes: widespread introductions and extirpations of narrow endemics lead to regional loss of dissimilarity. Divers Distrib. 2017; 23(2): 171–184. Publisher Full Text\n\nLiu C, Dudley KL, Xu ZH, et al.: Mountain metacommunities: climate and spatial connectivity shape ant diversity in a complex landscape. Ecography. 2018; 41(1): 101–112. Publisher Full Text\n\nWarsa A, Nastiti Krismono AS, Nurfiarini A, et al.: SUMBER DAYA PERIKANAN TANGKAP DI WADUK KOTO PANJANG, RIAU. Bawal. 2008; 2(3): 93–97. Publisher Full Text\n\nKrismono ASN, Lathifa R, Sukimin S: Food habits of Thynnichthys polylepis in Koto Panjang Reservoir, Riau. Jurnal lktiologi Indonesia. 2008; 1(8): 25–34. Reference Source\n\nAryani N: Native species in Kampar Kanan river, Riau Province Indonesia. Int J Fish Aquat Stud. 2015; 2(5): 213–217. Reference Source\n\nAryani N, Hasibuan S, Mardiah A, et al.: Morphometric Characteristics of Asian Catfish, Hemibagrus wyckii (Bleeker, 1858) (Bagridae), from the Riau Province of Indonesia. Pak J Biol Sci. 2017; 20(8): 382–389. PubMed Abstract \| Publisher Full Text\n\nPiria M, Simonovic P, Zanella D, et al.: Long-term analysis of fish assemblage structure in the middle section of the Sava River- The impact of pollution, ﬂood protection and dam construction. Sci Total Environ. 2019; 651(Pt 1): 143–153. PubMed Abstract \| Publisher Full Text\n\nKalogianni E, Vourka A, Karaouzas I, et al.: Combined effects of water stress and pollution on macroinvertebrate and fish assemblages in a Mediterranean intermittent river. Sci Total Environ. 2017; 603–604: 639–650. PubMed Abstract \| Publisher Full Text\n\nZhang C, Ding L, Ding C, et al.: Responses of species and phylogenetic diversity of fish communities in the Lancang River to hydropower development and exotic invasions. Ecol Indic. 2018; 90: 261–279. Publisher Full Text\n\nBouska KL, Houser JN, De Jager NR, et al.: Applying concepts of general resilience to large river ecosystems: A case study from the Upper Mississippi and Illinois rivers. Ecol Indic. 2019; 101: 1094–1110. Publisher Full Text\n\nTorres da Silva M, Pereira JO, Vieira LJS, et al.: Hydrological seasonality of the river affecting fish community structure of oxbow lakes: A limnological approach on the Amapá Lake, southwestern Amazon. Limnologica. 2013; 43(2): 79–80. Publisher Full Text\n\nFithra RY, Siregar YI: Fishes biodiversity of Kampar river an inventory from Kampar Kanan river. Journal of Environmental Science. 2010; 2(4): 139–147. Reference Source\n\nSimanjuntak CPH, Rahardjo MF, Sukimin S: Ichthyofauna in floodplain of Kampar Kiri river. Jurnal Ikhtiologi Indonesia. 2006; 6(2): 99–109. Reference Source\n\nNurdawati S, Muflikhah N, Sunarno MTD: Fisheries resources in the waters of the Batanghari river at Jambi. Bawal. 2006; 1(1): 1–10. Publisher Full Text\n\nBahri S: The observation of fish species in the waters of the Musi River in South Sumatra-Indonesia. Buletin Teknik Liktayasa Sumber Daya dan Penangkapan. 2007; 5(1): 1–4. Publisher Full Text\n\nKottelat M, Whitten T: Freshwater biodiversity in Asia LTith special reference to fish. The World Bank. Washington D.C. 1996. Reference Source" }	[ { "id": "52476", "date": "02 Sep 2019", "name": "Rudy Agung Nugroho", "expertise": [ "Reviewer Expertise My area research is about fish biology", "animal physiology", "fish nutrition", "and fish conservation." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn general this project is useful to add important information regarding fish fauna in Indonesia. However, some part in the article, need to be clarified by the author(s). Please see comments below:\nAbstract Conclusion: Nile tilapia should be mentioned as exotic species which found in upstream, cause it is a dominant species.\n\nIntroduction\n\"In 2015, the total ﬁshery production of Indonesia was 16,954,344 metric tons, of which 455,270 metrics tons were obtained from inland capture ﬁsheries, 6,065,060 metric tons were obtained from marine fisheries, and 10,074,014 metric tons were obtained from aquaculture fisheries production\". Use newest statistic data would be better, instead of 2015.\nMethods\nThough endangered species were returned to the river. Please explain on how the author caught this fish? Doesn’t hurt the fish?\n\nPlease also explain on how fish euthanization? Does all fish same treatment using piercing part on their brain?\n\nIt is stated in the methods that \"the length, body weight and morphometric characteristics were only collected for 10 individual specimens from each species. This mean in total 10 individual specimens from over a year? Please explain, because there are some species that have number less than 10 each month, for example Hemibagrus wyckii.\nResults\nThe name Pangasius hypophthalmus in the Table 2, should be changed to Pangasianodon hypophthalmus. Please check fishbase.org here.\n\nPlease also check the fish name for Pangasius pangasius.\nOverall, this article is well written and has good structure.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4907", "date": "09 Sep 2019", "name": "Netti Aryani", "role": "Author Response", "response": "Response for Rudy Agung Nugroho Comments:Abstract : We would like to clarify that “Nile tilapia as exotic species have been mention in the point of results.Introduction : the latest data we have in the year 2015, there is no recent data have been found from Indonesian Fisheries Statistics.Methods : We caught the fish using fishing pole. We have explained in the methods We have been explained in the Methods, yes…all fish are same treatment except for endangered species. Fish caught more than 10 individuals for a year, morphometric measurement for only 10 individual of fish. However, fish caught less than 10 individual, the morphometric analysis were measured for all fish. Results :1. We have been check in the fish base. The name of Pangasius pangasius is correct.2. The name of Pangasius pangasius is correct. Fishbase.org said that Pangasius pangasius spesies for aquaculture in Indonesia, Thailand, and Malaysia. However, Pangasianodon hypophthalmus species was categorize as endangered species and found in Mekong, Chao Phraya, and Maeklong basins." } ] }, { "id": "56362", "date": "25 Nov 2019", "name": "Christopher Marlowe Caipang", "expertise": [ "Reviewer Expertise Aquaculture", "Aquatic biotechnology", "health management" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript presented the results in a straightforward manner. The Introduction should contain studies that had been conducted in the area and the research gap that the authors need to address. Perhaps the authors could have done statistical analyses to compare the diversity in the sites where the data have been obtained. In the Discussion section, the authors must clearly explain the implications of their study and the impacts of the results that they had in terms of managing the resources. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5061", "date": "05 Feb 2020", "name": "Netti Aryani", "role": "Author Response", "response": "Response for Christopher Marlowe A. Caipang Comments:Introduction: We have been added new references in introduction who conducted research in this area.Methods: We do not use statistical analysis for calculate the data. All the data was entered into excel 2010 (version 14.0). Then, the data of different species for abundance and occurrence was calculated based on the equation of Shannon diversity Index, Simpson diversity index and Sorenson’s coefficient (CC)Discussion: we have been added the implication of our study and effect to managing fish resources. Such as the implication of exotic species for examples Nile tilapia and common carp have negative effect to native species in the upstream area of Koto Panjang Reservoir." } ] } ]	1	https://f1000research.com/articles/8-1435
https://f1000research.com/articles/9-86/v1	05 Feb 20	{ "type": "Systematic Review", "title": "A systematic review of non-randomised evaluations of strategies to improve participant recruitment to randomised controlled trials", "authors": [ "Heidi R Gardner", "Loai Albarquoni", "Adel El Feky", "Katie Gillies", "Shaun Treweek", "Loai Albarquoni", "Adel El Feky", "Katie Gillies", "Shaun Treweek" ], "abstract": "Background: Recruitment to trials can be challenging. Currently, non-randomised evaluations of trial recruitment interventions are rejected due to poor methodological quality, but systematic assessment of this substantial body of work may inform trialists’ decision-making about recruitment methods. Our objective was to quantify the effects of strategies to improve participant recruitment to randomised trials evaluated using non-randomised study designs. Methods: We searched relevant databases for non-randomised studies that included two or more interventions evaluating recruitment to trials. Two reviewers screened abstracts and full texts for eligible studies, then extracted data on: recruitment intervention, setting, participant characteristics, number of participants in intervention and comparator groups. The ROBINS-I tool was used to assess risk of bias. The primary outcome was the number of recruits to a trial. Results: We identified 92 studies for inclusion; 90 studies aimed to improve the recruitment of participants, one aimed to improve the recruitment of GP practices, and one aimed to improve recruitment of GPs. Of the 92 included studies, 20 were at high risk of bias due to confounding; the remaining 72 were at high risk of bias due to confounding and at least one other category of the ROBINS-I tool. The 20 studies at least risk of bias were synthesised narratively based on seven broad categories; Face to face recruitment initiatives, postal invitations and responses, language adaptations, randomisation methods, trial awareness strategies aimed at the recruitee, trial awareness strategies aimed at the recruiter, and use of networks and databases. The utility of included studies is substantially limited due to small sample sizes, inadequate reporting, and a lack of coordination around deciding what to evaluate and how. Conclusions: Careful thought around planning, conduct, and reporting of non-randomised evaluations of recruitment interventions is required to prevent future non-randomised studies contributing to research waste. Registration: PROSPERO CRD42016037718", "keywords": [ "Clinical trial", "Participant recruitment", "Recruitment interventions", "Participant selection", "Recruitment strategies" ], "content": "Abbreviations\n\nCINAHL, Cumulative Index to Nursing and Allie Health Literature; CONSORT, Consolidated Standards of Reporting Trials; EMBASE, Excerpta Medica database; GRADE, Grading of Recommendations Assessment, Development and Evaluation; MEDLINE, Medical Literature Analysis and Retrieval System Online; MRC, Medical Research Council; PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses; RCT, randomised controlled trial; ROBINS-I, Risk Of Bias in Non-randomised Studies – of Interventions; SWAT, study within a trial\n\n\nIntroduction\n\nRandomised controlled trials (RCTs) are at the core of evidence-based healthcare. They use random assignments to allocate participants to treatment groups, and therefore guard against selection bias1, whether these involve medicinal products, devices or services. Recruiting participants can be difficult, as can the process of recruiting clinicians to work on the trial with and on behalf of the trial team2.\n\nOne important source of evidence for trialists looking for rigorously evaluated evidence on how to effectively recruit participants to trials is the 2018 Cochrane systematic review of interventions to improve trial recruitment3. Despite having no date or language restrictions and including 72 recruitment comparisons, just three are supported by high-certainty evidence3.\n\nThis systematic review reported here uses a similar process to the 2018 Cochrane systematic review of interventions to improve trial recruitment3, but with one substantial difference. This review focusses only on recruitment interventions that are evaluated using non-randomised methods. Until now, systematic reviews of non-randomised studies of recruitment interventions have been scarcely undertaken due to the perception that non-randomised studies are individually, of low methodological quality. However, the systematic evaluation of a substantial amount of research activity is necessary and worthwhile; without collation, this body of evidence is currently being ignored, and may hold substantial/promising undiscovered effects. Whether evidence of benefit is found for one or more interventions, the trials community will benefit from knowing the outcome of this review. Moreover, aggregating data from non-randomised studies using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach4, may raise confidence in the overall body of evidence, and supplement the evidence-base from randomised studies.\n\n\nObjective\n\nWe conducted a systematic review of non-randomised studies that evaluated the effects of strategies to improve recruitment of participants to RCTs.\n\n\nMethods\n\nThe full protocol for this review has been previously published5 and registered with PROSPERO (CRD42016037718). No amendments have been made to the protocol since its publication. A brief summary of methods is given below.\n\nNon-randomised studies of two or more interventions to improve recruitment to a randomised trial. ‘Non-randomised studies’ are defined as any quantitative assessment of a recruitment intervention that did not randomly allocate participants to intervention or comparison groups. No additional eligibility criteria (e.g. publication year, status, language or journal) were applied.\n\nIndividuals enrolled in a trial. The context of the trial is likely to be healthcare but may not be, for the reason that interventions that are effective in other fields may also be applicable to settings in the healthcare environment.\n\nAny intervention or approach aimed at improving or supporting recruitment of participants nested within studies performed for purposed unrelated to recruitment.\n\nPrimary: Number of individuals or centres recruited into a trial.\n\nSecondary: Cost of using the recruitment intervention per trial participant.\n\nWe searched the following electronic databases without language restriction for eligible studies: Cochrane Methodology Register (CMR), Medical Literature Analysis and Retrieval System Online (MEDLINE), MEDLINE In-Process, Excerpta Medica dataBASE (EMBASE), Cumulative Index to Nursing and Allied Health Literature (CINAHL), and PsycINFO. The full search strategy is published and freely accessible5. Reference lists of relevant systematic reviews (e.g. 3) and included studies were hand-searched.\n\nThe literature searches were carried out between 16th October and 11th November 2015. On 2nd August 2018 an updated search was made in all databases, and a further 2,521 abstracts were found. 460 abstracts from 2018 were screened in duplicate, which led to 10 full texts being checked for inclusion. The ten full texts detailed ten studies, none of which provided sufficient detail about the design or implementation of interventions to allow us to pool data. Adding these studies into the review would not strengthen or disprove the conclusions we had already drawn. For this reason, we have chosen not to carry out a full updated search, and all data presented in this paper reflect the full literature searches carried out in 2015.\n\nTwo reviewers (HRG and one other) independently screened the abstracts of all search records. Full texts of potentially eligible abstracts were then independently reviewed by HRG and one other to determine inclusion. Disagreements were resolved through discussion.\n\nSearch results were merged, duplicate records removed, and a master spreadsheet was used to track all inclusions/exclusions to allow us to create a Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram (Figure 1). Data were extracted by two reviewers independently (HRG and one other) and collected on specially designed forms (Extended Data File 1 Blank data extraction form 6). Disparities were resolved through discussion.\n\nTwo members of the project team used the ROBINS-I tool7 to assess studies for aspects of methodological quality such as confounding, participant selection, intervention measurement, departures from the intended intervention, missing data, outcome measurement and selection of the reported result. As per ROBINS-I guidance, studies at critical risk of bias were excluded from any synthesis.\n\nStudies were analysed according to the type of intervention used; interventions were grouped when their form or content was deemed sufficiently alike. We planned to further categorise studies by participant if we found the same intervention applied to more than one type of participant (e.g. patients, staff at recruiting centres).\n\nAttempts were made to contact study authors to obtain missing data. Analyses were conducted on an intention-to-treat basis where possible; alternatively, data were analysed as reported.\n\nThe nature of the included studies meant that much of the analysis was anticipated to be narrative. Where population, intervention and outcome were sufficiently similar to allow for a meta-analysis, we planned to look for visual evidence of heterogeneity in forest plots, and statistical evidence of heterogeneity using the chi-square test for heterogeneity and the degree of heterogeneity quantified using the I2 statistic9. Where substantial heterogeneity was detected (I2 ≥ 50 %), we planned to investigate possible explanations informally and summarise data using a random-effects analysis where appropriate.\n\nWe planned to investigate reporting (publication) bias for the primary outcome using a funnel plot where 10 or more studies of the same population, intervention and outcome were available.\n\n\nResults\n\nWe screened a total of 9,642 abstracts identified by the database search, and 231 articles found through hand searching of review article reference lists. Of the screened abstracts, 256 were suitable to assess for inclusion at full-text stage. We were unable to obtain the full text of 33 of the 256 articles (details in Extended Data File 2 References to studies awaiting assessment6). Of the 223 full-text articles assessed, 124 were excluded; this includes seven articles which required additional data to allow for inclusion (details of excluded studies in Extended Data File 3 Characteristics of excluded studies6). A total of 99 full texts were included, which comprised 102 individual studies; 92 of these were considered to be at serious risk of bias while ten were considered to be at critical risk of bias. The latter group were excluded from the study as per ROBINS-I guidance (see risk of bias assessments for these studies in Extended Data File 4 Studies that were at a critical risk of bias and therefore excluded from this review6).\n\nOf the 92 included studies, 90 studies assessed interventions that aimed to improve the recruitment of participants to trials (55123 individuals and 172 couples), one assessed an intervention that aimed to improve the recruitment of GP practices to trials (54 practices), and one assessed an intervention that aimed to improve recruitment of GPs (150 GPs). 23 studies reported data on cost per recruit. Study size ranged between 14 and 5887 participants.\n\nThe design of included studies varied substantially and did not always fit into conventional design categories. Most studies (82/92) were what we describe as ‘yield’ studies. These types of studies appear not to have been planned as a method aiming to rigorously evaluate a recruitment intervention or interventions; rather, authors retrospectively report what methods have been used to recruit participants into the trial. Reporting of yield studies tends to rely on self-report by participants, although where online methods were used, the calculation of participant yield was recorded by the software or website; e.g. via number of clicks recorded by Facebook.\n\nThe remaining study types included in this review included cohort (7/92) and before and after designs (3/92).\n\nOf the 92 included studies, 72 were classified as at a ‘moderate’ or ‘serious’ risk of bias in one of more domains as well as the bias due to confounding domain. The remaining 20 studies were at ‘serious’ risk of bias in the confounding domain but were deemed to be at ‘low’ risk of bias across all other domains (participant selection, intervention classification, deviations from intervention, missing data, outcome measurement, selection of reported result).\n\nWe made the decision to focus on the 20 studies at least risk of bias in the Results and Discussion sections of this review. Primarily, this reflects our confidence in the evidence presented and follows a similar approach taken in the 2018 Cochrane recruitment review3. Data from the 72 studies that we have chosen not to focus on are presented in Extended Data File 5 Characteristics of included studies6. The 20 studies that we focus on here are organised into seven broad intervention categories (full references to these studies are presented in Extended Data File 6 References for the 20 studies included in the results section of this review6).\n\nThe seven intervention categories are:\n\nFace to face recruitment initiatives\n\nPostal invitations and responses\n\nLanguage adaptations\n\nRandomisation methods\n\nTrial awareness strategies aimed at the recruitee\n\nTrial awareness strategies aimed at the recruiter\n\nUse of networks and databases\n\nDue to the nature of the studies included in this synthesis, the above intervention categories frequently overlap to some extent within one study. Where lines between categories were not clear or distinct, we placed studies according to the emphasis given by the original study authors.\n\nTable 1 shows the number of participants, GPs, and practices recruited as a result of various interventions across the seven categories for the 20 studies. This table should be viewed with caution because the general lack of denominators means that direct and meaningful comparison within and across categories is not possible, as described below.\n\nLetters, e.g. ‘A’, refer to more than one intervention tested within the same intervention category. Numbers, e.g. ‘B1’, refer to instances where the same intervention was tested more than once within a study.\n\nComparing data within categories. The details of the interventions evaluated in these studies are limited, so to bring order to the variety of interventions, we have assigned them to broad categories. Each of these categories includes a range of interventions, the majority of which we are unable to thoroughly describe. For this reason, we urge you not to compare data within categories. By this we mean looking at two studies, e.g. Andersen 2010 and Bell-Syer 2000, seeing that 87 out of 187 participants and 104 out of 187 participants were recruited using face to face recruitment initiatives respectively, and assuming that these values demonstrate the success or failure of specific face to face recruitment initiatives.\n\nWe have simply used categories to bring order to the variety of interventions included in this review; each category includes a diverse range of interventions. This diversity in interventions means that none of the data presented have been pooled, and it is important that caution is exerted when interpreting data to ensure that we do not assign influence to studies where they are not deserving of it.\n\nComparing data across categories. Similarly, we urge you not to compare data across categories. By this, we mean looking at a study, e.g. Andersen 2010, seeing the 87 participants were recruited using face to face recruitment initiatives, and 100 participants were recruited using postal invitations and responses, and making a judgement about the success (or failure) of either of the interventions used. It’s important to bear in mind that these data do not provide denominators; there is no way for us to know how many people were exposed to either of these interventions, or over what time period, in order to recruit 187 participants.\n\nTen studies (totalling 3853 participants and 150 GPs) evaluated face to face recruitment initiatives, two of which used cohort studies, and eight used yield studies (see Table 1 and Table 2).\n\nFace to face recruitment initiatives varied across the ten studies in this category; largely they focussed on recruitment of participants who were attending appointments with their primary care physician or GP, other studies looked at recruiting participants who were in the waiting room of their care-provider before an appointment took place. In most cases, waiting room recruitment was facilitated by a research nurse. Other methods used include referral of participants from different parts of their own clinical care pathway, though most were targeted around an existing appointment made by the potential participant. These care pathways included outpatient appointments, appointments at community institutions, academic institutions, and at veterans’ health administration centres.\n\nDespite the superficial similarity of the interventions used within this category, both the diversity of comparators, settings and populations, and the poor reporting of the specifics of the interventions, made pooling data unfeasible.\n\nOne study (2575 participants) evaluated language adaptations; Barrera 2014 compared translations of Google AdWords in Spanish or English language using a yield study (see Table 1 and Table 2). The trial was based online within the USA and aimed to recruit pregnant women to a trial of an internet intervention for postpartum depression, the embedded recruitment study did not account for variations in how common postpartum depression is in Spanish-speaking populations in comparison to English-speaking populations.\n\nNine studies (totalling 1614 participants) evaluated postal invitations and responses, two of which used cohort studies, and the other seven used yield studies (see Table 1 and Table 2).\n\nPostal invitations and responses were used widely within the studies included in this review. Largely interventions within this category were based on patient lists held by caregivers; letters were sent out and then the number of responses from potential participants monitored, in most cases these studies reported the number of responses from people that ultimately went on to be recruited into the study. As mentioned in the ‘face to face recruitment initiatives’ section, many of the postal interventions used a face to face method as their comparator. Despite the superficial similarity of the interventions used within this category, both the diversity of their comparators, settings and populations, and the poor reporting of the specifics of the interventions, made pooling data unfeasible. In only one case (Funk 2010), did comparators vary from this trend. In this study, the method of response to a mailed brochure was monitored; potential participants were given the option of responding to the mailing by telephone or a website. These comparators were unusual within the literature, and draw attention to the two-dimensional nature of many of the other studies within this category; largely researchers looking at postal methods are focussing on the method used to contact potential participants, rather than the ways that these individuals may respond.\n\nOne study (553 participants) evaluated randomisation methods; Brealey 2007 compared use of telephone and postal randomisation methods using a yield study (see Table 1 and Table 2). Initially, general practices involved used a telephone service to randomise patients to the host trial. Delays in the start of recruitment at some sites led the team to modify the randomisation procedure to include postal randomisation. Following this, new sites were given the option to use either postal or telephone randomisation methods.\n\nFour studies (totalling 407 participants) evaluated trial awareness strategies aimed at the recruitee, one of which used a before and after study, and the remaining three used yield studies (see Table 1 and Table 2).\n\nThis category is diverse; the four studies include four distinct interventions. The reporting of these interventions is ambiguous; for example, Carr 2010 describes a community outreach event, Johnson 2015 describes a non-targeted flyer, and Sawhney 2014 describes increased awareness of the trial via use of a telephone reminder prior to their clinic appointment. It is feasible that all of these interventions could come under the umbrella of ‘trial awareness strategies aimed at the recruitee’ which is what is described by Carter 2015. The text states that Carter 2015’s interventions included distribution of leaflets and posters at clinics, therapy centres and regional multiple sclerosis societies, presentations and attendance at regional multiple sclerosis events and to local physiotherapy teams, and referral from other professional such as multiple sclerosis nurses and word of mouth.\n\nFives studies (totalling 188 participants and 54 practices) evaluated trial awareness strategies aimed at the recruiter, one of which used a cohort study, two used before and after studies, and two used yield studies (see Table 1 and Table 2).\n\nAgain, the interventions evaluated within this category are diverse: Carr 2010 looked at a medical education event; Embi 2005 and Treweek 2010 looked at methods of clinical trial alert software set to trigger during clinic appointments; Beauharnais 2012 assessed effectiveness of an automated pre-screening algorithm to identify potential participants; and Colwell 2012 evaluated the use of viral marketing techniques in the form of postcards, invitation letters and flyers. The diversity of these interventions means that data could not be pooled.\n\nTwo studies (totalling 486 participants) evaluated the use of networks and databases, both of which used yield studies (see Table 1 and Table 2).\n\nPark 2007 compared centralised recruitment efforts with de-centralised approaches that were tailored to the study and sites specifically. Weng 2010 evaluated effectiveness of existing lists of potentially eligible participants; comparing a clinical patient registry with a clinical data warehouse. The interventions are sufficiently different that data could not be pooled.\n\n\nDiscussion\n\nThis review identified 92 studies, 20 of which were included in a narrative synthesis; those 20 studies evaluated the effect of seven categories of interventions to improve recruitment to randomised trials.\n\nThe interventions evaluated in these studies varied significantly; even those that had an intervention category in common were sufficiently dissimilar to prevent pooling of data, rendering sub-group analyses unfeasible. That said, what limits the utility of these studies is not necessarily the interventions evaluated; it is the abundance of small study samples sizes, inadequate reporting, and a lack of coordination when it comes to deciding what to evaluate and how.\n\nThis review does not show ground-breaking evidence that will change the global landscape of how trialists recruit participants into trials. However, the 2018 Cochrane recruitment review3 of randomised evaluations of recruitment interventions was not able to provide clear evidence of benefit for the majority of interventions either. Like this review, the randomised review also experienced challenges with small, methodologically flawed studies, a diverse range of interventions, and a lack of detailed reporting. This fact may not be comforting for trialists, but it demonstrates that the utility of non-randomised studies is not always vastly different from their randomised counterparts.\n\nNon-randomised evaluations have acquired a bad reputation, but they do have their merits. Randomised evaluations are not always possible because of logistics, financial resources, or ethical reasons10, and non-randomised studies could allow researchers to gather useful data to complement or replace data generated by randomised trials11.\n\nIt is clear that non-randomised evaluations of recruitment interventions will continue. In their current form, however, we found their usefulness to others to be extremely limited. What we need to focus on now is improving the way that these non-randomised evaluations are planned, conducted and reported.\n\nThe non-randomised studies that are included in this review largely take the form of what we refer to as ‘yield’ studies. As described earlier, these types of studies appear not to have been planned as a means to rigorously evaluate a recruitment method; instead, they represent the work of authors retrospectively reporting what they have done, and subsequently what they have seen.\n\nThis practice limits utility of these studies in two ways:\n\n1. The studies are not designed in such a way as to lend themselves to straightforward analysis, which means that interventions and their comparator are not always introduced at the same time or used for the same length of time. A lack of planning also results in the collection of data that are incomplete and lack context; this is a problem that features in most studies included in this review. Data are presented in terms of numerators; they provide numbers of participants/GPs/practices recruited into a trial, but do not provide a denominator, meaning that comparing interventions to assess effectiveness is impossible.\n\n2. As is clear from the larger intervention categories such as face to face recruitment initiatives, and postal invitations and responses, the trials community is currently lacking a consistent approach to the non-randomised evaluations that they are publishing.\n\nRather than reporting what has been done retrospectively, we would encourage trialists to prospectively plan to embed recruitment evaluations, specifically using a study within a trial (SWAT) protocol12 that already exists on the SWAT repository13, into their trials from the very beginning of the process of planning the host trial. The Medical Research Council Systematic Techniques for Assisting Recruitment to Trials (START) project is a remarkable example of the effectiveness of a well-planned, organised and cohesive approach to SWATs14; the project ran between 2009 and 2015 and answered its research question regarding optimised participant information sheets within the space of six years. The follow-on PROMETHEUS project is co-ordinating over 30 recruitment and retention SWATs and will substantially increase global evidence for trial recruitment and retention in the space of around four years. Without coordination of high-quality evaluations, it is entirely possible for a decade to pass without materially increasing the evidence base available to trialists, as a comparison of the 200715 and 20183 Cochrane recruitment reviews demonstrates.\n\nThe process of conducting non-randomised evaluations of recruitment lacks structure; limited planning means that many of the studies included in this review were penalised as a result of poor conduct.\n\n74% of included studies were judged to be at moderate risk of bias in the ‘bias in classification of interventions’ domain of the ROBINS-I tool. These studies were most often penalised as a result of blurred lines between interventions and their comparators. For example, Adams 1997 (Extended Data File 5 Characteristics of included studies6) compares the effectiveness of professional referrals, cold calling by the research team, presentations at senior centres, media outreach, mailings sent to personal care home managers, and flyers; a total of six interventions. Participants could conceivably have been drawn to take part in the trial as a result of more than one of these six interventions; someone could have seen the media outreach campaign, received a flyer, and attended a presentation at a senior centre. This, combined with self-report of one method by participants, makes meaningful interpretation of the results extremely difficult.\n\nCurrently, trialists are focussing on the mode of delivery of the interventions that they are working to evaluate; they omit key details regarding the content of the intervention, as well as the specific timescales that interventions were in place for. We highly encourage the use of the Guidelines for Reporting Non-Randomised Studies16, and the Template for Intervention Description and Replication (TIDieR) checklist and guide17 when reporting these types of studies.\n\nMissing data was another aspect of reporting where detail was lacking. Of the 92 included studies, 13% were deemed to be at serious risk of bias due to missing data; a glaring example of research waste. Pieces of data that were missing were not entire data categories or a reflection of participants being lost to follow-up; in some cases, the data simply did not add up. One example is Blackwell 2011; this paper reported recruitment of 301 participants, but when we manually calculated how many participants had been recruited across each of the seven methods used in the study, and also included the participants that were reported as ‘don’t know/refused/other’, the total was 303 participants. The size of the discrepancy may appear trivial, but it undermines confidence in the data presented and the study generally. This was not a unique occurrence; missing data were also found in Brownstone 201218, Freret 200319, Kernan 200920, Lewis 199821, Martin 201122, McDermott 200923, Piantadosi 201524, Silagy 199125, Tenorio 201126, Unlü Ince 201427, and Zhou 201328. If non-randomised evaluations of recruitment interventions are to have any value, how they are reported needs to improve.\n\n\nConclusions\n\nSome interventions to increase recruitment described in this review do show promise but methodological and reporting problems mean that our confidence in these results is not substantial enough to recommend changes to current recruitment practice. Currently the literature is oversaturated with a diversity of interventions tested in non-randomised evaluations that fail to drill down deep into the effects of each specific recruitment strategy. Their usefulness to other trialists is therefore extremely limited.\n\nWhat is needed now is a move away from retrospective descriptions of what happened, to carefully planned prospective evaluations of well-described recruitment interventions and their comparators. Without this change, authors of non-randomised evaluations of recruitment interventions are simply contributing to research waste.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nOpen Science Framework: A systematic review of non-randomised evaluations of strategies to improve recruitment to randomised controlled trials. https://doi.org/10.17605/OSF.IO/98BQ46\n\nThis project contains the following extended data:\n\n- Extended Data File 1 Blank data extraction form.docx\n\n- Extended Data File 2 References to studies awaiting assessment.docx\n\n- Extended Data File 3 Characteristics of excluded studies.docx\n\n- Extended Data File 4 Studies that were at a critical risk of bias and therefore excluded from this review.docx\n\n- Extended Data File 5 Characteristics of included studies.docx\n\n- Extended Data File 6 References for the 20 studies included in the results section of this review.docx\n\nOpen Science Framework: A systematic review of non-randomised evaluations of strategies to improve recruitment to randomised controlled trials. https://doi.org/10.17605/OSF.IO/98BQ46\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe would like to thank Beverley Smith for her help with obtaining full-text articles, and Polly Black, Gordon Fernie, Kirsty Loudon, Sarah Stirrup, and Daniel Williams for their help with data extraction. Daniel Williams was supported by a summer internship with Medical Research Scotland.\n\n\nReferences\n\nOdgaard-Jensen J, Vist GE, Timmer A, et al.: Randomisation to protect against selection bias in healthcare trials. Cochrane Database Syst Rev. 2011; (4): MR000012. PubMed Abstract \| Publisher Full Text\n\nWalters SJ, Bonacho dos Anjos Henriques-Cadby I, Bortolami O, et al.: Recruitment and retention of participants in randomised controlled trials: a review of trials funded and published by the United Kingdom Health Technology Assessment Programme. BMJ Open. 2017; 7(3): e015276. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTreweek S, Pitkethly M, Cook J, et al.: Strategies to improve recruitment to randomised trials. Cochrane Database Syst Rev. 2018; 2: MR000013. PubMed Abstract \| Publisher Full Text\n\nGuyatt GH, Oxman AD, Vist GE, et al.: GRADE: an emerging consensus on rating quality of evidence and strength of recommendations. BMJ. 2008; 336(7650): 924–926. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGardner HR, Fraser C, MacLennan G, et al.: A protocol for a systematic review of non-randomised evaluations of strategies to improve participant recruitment to randomised controlled trials. Syst Rev. 2016; 5(1): 131. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGardner H: “A Systematic Review of Non-randomised Evaluations of Strategies to Improve Participant Recruitment to Randomised Controlled Trials.” OSF. 2020. http://www.doi.org/10.17605/OSF.IO/98BQ4\n\nSterne JA, Higgins JP, Elbers RG, et al.: Reference Source\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: the PRISMA Statement. Open Med. 2009; 3(3): e123–30. PubMed Abstract \| Free Full Text\n\nHiggins JP, Thompson SG, Deeks JJ, et al.: Measuring inconsistency in meta-analyses. BMJ. 2003; 327(7414): 557–560. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSibbald B, Roland M: Understanding controlled trials. Why are randomised controlled trials important? BMJ. 1998; 316(7126): 201. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchünemann HJ, Tugwell P, Reeves BC, et al.: Non-randomized studies as a source of complementary, sequential or replacement evidence for randomized controlled trials in systematic reviews on the effects of interventions. Res Synth Methods. 2013; 4(1): 49–62. PubMed Abstract \| Publisher Full Text\n\nTreweek S, Bevan S, Bower P, et al.: Trial Forge Guidance 1: What is a Study Within A Trial (SWAT)? Trials. 2018; 19(1): 139. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSWAT Repository. Reference Source\n\nRick J, Graffy J, Knapp P, et al.: Systematic techniques for assisting recruitment to trials (START): study protocol for embedded, randomized controlled trials. Trials. 2014; 15: 407. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\n2007 cochrane review.\n\nReeves BC, Gaus W: Guidelines for reporting non-randomised studies. Forsch Komp Klas Nat. 2004; 11(Suppl 1): 46–52. PubMed Abstract \| Publisher Full Text\n\nHoffmann TC, Glasziou PP, Boutron I, et al.: Better reporting of interventions: template for intervention description and replication (TIDieR) checklist and guide. BMJ. 2014; 348: g1687. PubMed Abstract \| Publisher Full Text\n\nBrownstone L, Anderson K, Beenhakker J, et al.: Recruitment and retention in an adolescent anorexia nervosa treatment trial. Int J Eat Disord. 2012; 45(6): 812–815. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFreret N, Ricci L, Murphy S: Recruiting and screening older, transitional to frail adults in congregate living facilities. Appl Nurs Res. 2003; 16(2): 118–125. PubMed Abstract \| Publisher Full Text\n\nKernan WN, Viscoli CM, DeMarco D, et al.: Boosting enrollment in neurology trials with Local Identification and Outreach Networks (LIONs). Neurology. 2009; 72(15): 1345–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLewis CE, George V, Fouad M, et al.: Recruitment strategies in the women's health trial: feasibility study in minority populations. WHT:FSMP Investigators Group. Women's Health Trial:Feasibility Study in Minority Populations. Control Clin Trials. 1998; 19(5): 461–476. PubMed Abstract \| Publisher Full Text\n\nMartin MA, Swider SM, Olinger T, et al.: Recruitment of Mexican American adults for an intensive diabetes intervention trial. Ethn Dis. 2011; 21(1): 7–12. PubMed Abstract \| Free Full Text\n\nMcDermott MM, Domanchuk K, Dyer A, et al.: Recruiting participants with peripheral arterial disease for clinical trials: experience from the Study to Improve Leg Circulation (SILC). J Vasc Surg. 2009; 49(3): 653–659.e4. PubMed Abstract \| Publisher Full Text\n\nPiantadosi C, Chapman IM, Naganathan V, et al.: Recruiting older people at nutritional risk for clinical trials: what have we learned? BMC Res Notes. 2015; 8: 151. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilagy CA, Campion K, McNeil JJ, et al.: Comparison of recruitment strategies for a large-scale clinical trial in the elderly. J Clin Epidemiol. 1991; 44(10): 1105–1114. PubMed Abstract \| Publisher Full Text\n\nTenorio SL, Gamito EJ, Ogden S, et al.: A special program to increase the participation of Hispanics in the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial. Hispanic Health Care Int. 2011; 9(1). Publisher Full Text\n\nÜnlü Ince B, Cuijpers P, van 't Hof E, et al.: Internet-based, culturally sensitive, problem-solving therapy for Turkish migrants with depression: randomized controlled trial. J Med Internet Res. 2013; 15(10): e227. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou ES, Dunsiger SI, Pinto BM: Proactive versus reactive recruitment to a physical activity intervention for breast cancer survivors: does it matter? Clin Trials. 2013; 10(4): 587–592. PubMed Abstract \| Publisher Full Text" }	[ { "id": "59580", "date": "13 Feb 2020", "name": "Karen Bracken", "expertise": [ "Reviewer Expertise Trial operations methodology research" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis systematic review is a companion to the 2018 Cochrane review of strategies to improve recruitment to clinical trials. Unlike the 2018 review, which included only randomised evaluations of strategies, this review includes only non-randomised evaluations. Given the 2018 review found that few recruitment strategies were supported by good quality evidence, this new review had the potential to build on the limited evidence base for trialists seeking strategies to improve their trial’s recruitment. The authors faced the challenge of synthesizing a large number of diverse evaluations and so the resulting synthesis is wide-ranging and necessarily high-level. The authors found the included studies of insufficient quality to allow for pooling of data and, as they point out, this limits the usefulness of this review in guiding trialists to select promising recruitment strategies. They mentioned in the conclusion that some interventions showed promise but it was unclear to me which specific interventions these were. This would be useful information to guide future research. The large number of retrospective, comparative “yield” studies included in the review is perhaps unsurprising given that many evaluations were seeking to compare strategies to invite people to take part in trials. The authors criticize these studies for reporting the numerator but not the denominator in such comparisons, for blurring lines between interventions/comparators and for lack of forward planning when designing evaluations. However, these limitations seem to reflect the real-world challenges of implementing such strategies. Even with forward planning, there seems to be no way to calculate the denominator for strategies like media outreach and flyers, and there may be few practical ways to prevent participants from being exposed to multiple strategies without causing potential harm to the host trial recruitment. Did the authors feel there was any way that such evaluations could be of value outside the trial in which they were implemented? The authors did suggest that recruitment evaluations would be of greater value if they reported their interventions more clearly and completely, using the TIDieR checklist. This seems like a practical suggestion and I hope it will be widely taken up. I found this review a thought-provoking but sobering read. The fact that such a large-scale, rigorously-conducted review resulted in not a single recommendation of a strategy to improve recruitment should be a wake-up call to all of us researching in this area.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] }, { "id": "65416", "date": "30 Jun 2020", "name": "Barbara Clyne", "expertise": [ "Reviewer Expertise Evidence synthesis", "RCTs", "process evaluation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis systematic review includes non-randomised studies of strategies to improve participant recruitment to randomised controlled trials. The justification for inclusion of non-randomised studies is that the previous Cochrane systematic review of RCTs found very little high-certainty evidence to guide trialists. This is a well presented, large scale, robust systematic review of a topic of great importance to trialists seeking strategies to improve their trial’s recruitment. The inclusion of non-randomised studies resulted in a diverse number of evaluations and the narrative synthesis is as a result quite broad and high-level. The recommendations for use of TIDIER in intervention reporting and SWATS are practical and feasible. There are a number of clarifications that I feel would improve the manuscript overall. These are as follows:\nIn the introduction could you please give some detail as to what strategies were identified as effective from the Cochrane review. In the discussion can you compare and contrast the findings of this current review to those of the Cochrane review. It would be helpful to get an understanding of what the addition of these non-randomised studies adds overall to the evidence base, given the substantial amount of work in this review. Included studies were of insufficient quality to allow for the pooling of data, however, the authors could present narratively a summary of strategies that demonstrated success, to guide trialists, and those designing SWATS as to what strategies may be worth exploring further.\n\nThe literature search was carried out in 2015. The authors have justified this by including details of how a search in 2018 identified an additional 10 studies only, the addition of which would not strengthen or disprove the conclusions. However, this search is 5 years out of date and I think the authors should contextualise their conclusions in light of this limitation. The addition of more ‘yield studies’ would be unlikely to change the conclusions (as these would be likely high risk of bias and not included in the analysis), however, the addition of a few more cohorts studies could have a bigger impact.\n\nWere all non-yield studies pre-planned studies?\n\nThe review identified 92 studies, but only 20 were considered low risk of bias and included in the narrative review. Could the authors comment on 20 included studies in more detail in the manuscript? For example, from my reading for the table, this includes 13 yield studies and 7 cohorts, etc. type studies.\n\nCould the authors comment on the definition of non-randomised study – I wonder if a more detailed definition was applied would this have reduced the number of yield studies that were identified?\n\nIn the introduction, the authors comment that aggregating data from non-randomised studies using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach, may raise confidence in the overall body of evidence, and supplement the evidence-base from randomised studies. The authors have not used GRADE in this manuscript or commented on why they have not. There is good recent guidance on the use of GRADE with ROBINS-I available, see Schünemann HJ, Cuello C, Akl EA, et al. GRADE guidelines: 18. How ROBINS-I and other tools to assess risk of bias in nonrandomized studies should be used to rate the certainty of a body of evidence. J Clin Epidemiol. 2019;111:105-114.1\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] }, { "id": "65427", "date": "03 Jul 2020", "name": "Emily Peckham", "expertise": [ "Reviewer Expertise Severe mental illness", "smoking cessation", "RCTs" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for inviting me to review this systematic review of non-randomised evaluations of strategies to improve participant recruitment to RCTs. I have listed some suggestions below that I believe would improve the article.\n\nSearch methods:\nWere there any date limits applied to the searches or were they from the databases inception?\n\nThe number of additional abstracts identified after the 2018 search is given in the methods but not the number identified in the original search which is confusing. I am also not convinced by this rationale for not updating the searches. I think it would be better to update the searches and include the information from the 10 studies identified. I am not clear why this wasn't done.\n\nData extraction:\nIt would be helpful to give an overview of what data was extracted.\n\nFigure 1:\nIt would be helpful to list the reasons for exclusion at the full-text stage.\n\nIt would be better to combine the included studies boxes into one, stating 'x articles were identified comprising of x unique studies' or similar\n\nResults:\nI am not sure what is meant by the statement that 7 articles were excluded at the full-text stage because they needed additional data to allow for inclusion. From the information in the study was it not possible to determine whether the study was eligible? Or is it that there was some of the data you wanted to extract was missing. If it is the latter I would include the study and state that there was missing data.\n\nUsing risk of bias to select studies:\nOf the 72 studies excluded what was their risk of bias due to the confounding domain, it just states 'the bias due to confounding domain'.\n\nComparing data within categories:\nI think the following statement needs to be rephrased or removed. I am not clear why you are unable to describe the intervention. 'Each of these categories includes a range of interventions, the majority of which we are unable to thoroughly describe.'\n\nPlanning no-randomised studies:\nThe acronym SWAT is used for the 1st time without being spelt out fully, it is only spelt out fully later on in the paragraph.\n\nIt would be useful in this section to explain what a SWAT is, as it stands the section focuses on what is limiting the utility of evaluations of recruitment and it would be improved and more useful if more focus is given on what could be done to increase the utility of these studies.\n\nReporting non-randomised studies:\nIt would be helpful to give an overview of the type of data that was missing. At present this section is focusing on the fact that the number of participants didn't add up in some of the studies. Was this the main example of missingness?\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] }, { "id": "65424", "date": "08 Jul 2020", "name": "Geoff K Frampton", "expertise": [ "Reviewer Expertise Evidence synthesis" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article reports on a systematic review of non-randomised studies that have evaluated approaches for improving the recruitment of people into randomised controlled trials (RCTs). The objective was “to quantify the effects of strategies to improve participant recruitment to randomised trials evaluated using non-randomised study designs”. The authors acknowledge that “currently, non-randomised evaluations of trial recruitment interventions are rejected due to poor methodological quality”, but their rationale for conducting this review is that “without collation, this body of evidence is currently being ignored, and may hold substantial/promising undiscovered effects”.\nThis is an important area of research since recruitment into RCTs is challenging; and failed or suboptimal trial recruitment often leads to trials being abandoned, creating a substantial amount of research waste, as well as having negative effects on trial participants and delaying the uptake of clinical trial findings into policy and practice.\n\nGeneral comments:\n\nI have read this paper with interest because most studies on trial recruitment are non-randomised and it would, therefore, be helpful to obtain as much useful information as possible from these studies to inform research practice and policy. However, a major obstacle, as the authors acknowledge, is the poor methodological quality of non-randomised recruitment studies. According to the stated rationale for doing this work I was expecting this systematic review to make the best use of the available data that could be extracted from these methodologically poor studies, thereby providing an advance on previous research. For instance, the methods section suggests that GRADE methods might enable methodologically weak studies to contribute informatively to data synthesis. But this review did not explore any methods such as GRADE that might have enabled any of the data from these studies to contribute to a comparative analysis. And the review stops short of providing any comparative analysis, thereby failing to address its stated objective. I assume that this is because the included studies were deemed too weak to be useful, although this is not explicitly stated or discussed. I agree with the authors that the statistical pooling of studies' results would not have been appropriate, due to the heterogeneity of study designs. A narrative synthesis would, therefore, be reasonable, as the authors acknowledge. However, no integration of the comparative results, strengths, and limitations of any of the individual studies is presented, meaning that effectively there is no narrative synthesis (though a summary of some study characteristics is given).\n\nI appreciate that it is not easy to do a systematic review on this topic area due to the considerable heterogeneity and weaknesses of the studies, so I sympathise with the authors. But I am surprised that some of the problems with this evidence base were not identified during the protocol development, scoping, and pilot testing of the methods. The upshot is that very weak (and seemingly useless) studies appear to have made it all the way through to the data synthesis step but then all of them were rejected from narrative synthesis. Could the evidence synthesis planning process have been done differently perhaps, to identify some of the methodological problems (e.g. limited sample sizes and ambiguous descriptions) earlier, or would this have just resulted in all studies being excluded? It feels to me that all studies have, effectively, been excluded anyway since none of them provide any comparative results to support the review objective. Are there any lessons that review teams can learn here, to improve future evidence syntheses in this difficult area?\n\nA comprehensive risk of bias assessment was conducted, but this is not reported consistently. For example, the 7 individual domains of bias are reported for the 10 studies that were excluded because they were at critical risk of bias (Extended data file 4) but only an overall risk of bias judgement is reported for the 72 studies that were included in the review (Extended data file 5). Given that a total of 92 studies were eligible for inclusion in the review, the risk of bias assessments for 10 studies appear to be missing (Extended data files 4 and 5 report 82 studies in total).\n\nThe review report is not fully clear about which of the methodological issues discussed were assessed as risks of bias, and which were identified additionally. Missing data are described separately in the Discussion (e.g. for the Blackwell 2011 study) but I presume that these missing data had already been captured in the “Missing data” domain of the risk of bias assessment? It would be helpful if each of the risk of bias domains could be explained so that their interpretation in relation to non-randomised recruitment studies is clear.\n\nIn summary, this review has failed to meet its stated objective and it is difficult to see how it advances knowledge in this scientific area (we already knew that non-randomised studies are methodologically weak, and the rationale behind this review was to make best use of these studies despite their limitations... which hasn't been achieved).\n\nBut I believe the authors could update this manuscript to make it into a useful scientific contribution. I have suggested two possibilities that the authors could consider. I am not sure how much effort these would take but they would utilise the studies already included in the review and may require little if any amendment to the current protocol:\nCheck the included studies and evaluate systematically whether there are any data that can be obtained from these non-randomised studies that would constructively inform a data synthesis (i.e. go back and attempt to fulfill the stated objective). Currently, the review considers only limitations, whereas it would be helpful to look for the strengths (if any) that can be taken from these non-randomised studies. Would GRADE or other approaches (e.g. sensitivity analyses) enable any of the included studies to contribute to data synthesis? Could you make best- / worst-case assumptions in sensitivity analyses to address some of the uncertainties? (see specific comments below). I would be surprised if all 92 identified studies are so poor that none of them at all can be descriptively reported in the narrative synthesis, but this should be systematically checked and transparently reported so that if it is true that the entire evidence base is useless then at least the conclusion would be defensible.\n\nIf the evidence-based conclusion is that data synthesis is not feasible due to all studies being too weak, then pragmatically the review could direct its effort to systematically analysing the limitations of the studies as a basis to support evidence-based recommendations on how to improve non-randomised studies in future. In order to make the critique of the studies’ methods more systematic, consistent, and transparent perhaps a table could be provided in the results section for all the included studies indicating exactly which methodological limitations each of the studies was susceptible to? This could then support evidence-based specific recommendations about exactly what needs to be improved in the non-randomised studies for research practice to be improved. I think this could make the paper valuable – provided that the recommendations arising are evidence-based and are communicated and disseminated carefully to achieve impact.\n\nSpecific comments:\nMethods: Types of intervention: I don’t understand the meaning of the following sentence (NB minor typo needs correcting): “Any intervention or approach aimed at improving or supporting recruitment of participants nested within studies performed for purposed unrelated to recruitment.”\n\nMethods: Search methods for identification of studies: “Adding these studies into the review would not strengthen or disprove the conclusions we had already drawn.” This statement seems to imply that the 10 studies being referred to were somehow tested for their influence on the overall results. I suspect this is not the intended meaning. Do you mean that, by virtue of their lack of clarity, these studies could not usefully inform the review?\n\nMethods: Assessment of risk of bias in individual studies: Very little description or discussion of the risk of bias assessment is provided. As ROBINS-I is a relatively new tool I wonder how many readers will be familiar with it? Could a supporting explanation be provided of how decisions were reached? If the review is re-framed to focus on the studies’ weaknesses (see my general comments above), then a more detailed discussion of risks of bias would be appropriate. How good was reviewer agreement on rating the risks of bias? Were any specific bias domains particularly difficult to interpret or agree on?\n\nFigure 1: PRISMA chart: What were the reasons that so many (33) full-text articles were not available? Is there anything practical that could be recommended to reduce this problem in future research?\n\nResults: Screening and identification of studies: “this includes seven articles which required additional data to allow for inclusion”. The meaning of this statement is not clear. Do you mean that insufficient information was reported in these studies to make an eligibility decision and since the authors did not provide any further information these studies had to be excluded?\n\nResults: Using risk of bias to select studies: The meanings of the seven bullets listed here are not explicitly defined anywhere, although to me they seem intuitive and self-evident. An exception is “Language adaptations” which I didn’t understand the meaning of until I reached Table 2, where I noticed that it is explained (implicitly). Perhaps cross-refer the reader to Table 2 to clarify what these bullets mean?\n\nTable 8: Minor typo in the heading of Column 8.\n\nResults: Postal invitations and responses: “As mentioned in the ‘face to face recruitment initiatives’ section, many of the postal interventions used a face to face method as their comparator”. This statement might be misleading as the text in the Face to face recruitment initiatives section does not say anything about postal interventions. However, the information referred to is in Table 2, so perhaps refer directly to the table? For studies that spanned these two categories, how was a decision made on which category to allocate the study to?\n\nResults: Trial awareness strategies aimed at the recruitee: Minor typo: “professional”.\n\nResults: Trial awareness strategies aimed at the recruiter: Minor typo: “fives”.\n\nDiscussion: “Non-randomised evaluations have acquired a bad reputation, but they do have their merits. Randomised evaluations are not always possible because of logistics, financial resources, or ethical reasons” This statement seems to be referring to non-randomised studies in general. Are there specific reasons why some (or indeed most) types of recruitment study can only be non-randomised?\n\nDiscussion: Some information about the Adams 1997 study is revealed here but it feels like this information should have been reported in the results section, along with equivalent information from the other included studies, for consistency and transparency.\n\nDiscussion: “Currently, trialists are focussing on the mode of delivery of the interventions that they are working to evaluate; they omit key details regarding the content of the intervention, as well as the specific timescales that interventions were in place for.” Is this statement evidence-based? It doesn’t really link to the results section.\n\nDiscussion: The issue of missing data has not been consistently covered for each study in the results section, so it is unclear how representative the discussion of missing data in the Blackwell 2011 study is of the studies in general. It feels harsh to say that 2 missing data (<1%) would be sufficient to cause mistrust in the entire Blackwell 2011 study. Were such strict criteria applied across all studies? Could you do a (quantitative or descriptive) sensitivity analysis – i.e. explore whether changing the sample size by n=2 would make any tangible difference to the outcomes? I think even Cochrane Review guidance is pragmatic about not being overly strict if the amount of missing data is trivial?\n\nDiscussion: The review included several study designs of which Yield studies were the most frequent and, apparently, least useful. Could the remaining study types that would be more reliable tell us anything? No results from any of these studies have been provided.\n\nConclusions: The statement “Some interventions to increase recruitment described in this review do show promise” is not evidence-based, since no evaluation of intervention effectiveness has been provided.\n\nReferences: There is no information provided for reference 15.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [] }, { "id": "65425", "date": "10 Jul 2020", "name": "Lucy Beasant", "expertise": [ "Reviewer Expertise Qualitative Researcher: Randomised Controlled Trials", "Equipoise", "Recruitment and Retention." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for requesting that I review this interesting and timely article, I found it very informative. Interventions that enhance recruitment to non-randomised research studies are an important and under reported area, this systematic review highlights the lack of clear, reliable evidence presented to date. This is an important article because it highlights methodological and reporting problems, whilst at the same time bringing together evidence from interventions used in non-randomised studies that do show ‘promise’ in terms of improving recruitment. In addition the article discusses and highlights the way in which future non-randomised studies might improve reporting (and conduct) of similar recruitment interventions, enabling researchers to investigate and utilise effective recruitment strategies during the planning and implementation stages of research studies. In my view this systematic review has been conducted with methodological rigour, however, I am a qualitative researcher/trialist and trust that this article will also be reviewed by a statistician/ systematic reviewer who routinely uses the ROBINS-I tool to assess for risk of bias. The authors have provided clear and explicit detail in relation to the studies and intervention categorisation used for the narrative synthesis of data. I believe this is a very strong piece of work in its current format and would recommend indexing.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-86
https://f1000research.com/articles/9-85/v1	05 Feb 20	{ "type": "Brief Report", "title": "Solubility and stability of melatonin in propylene glycol, glycofurol, and dimethyl sulfoxide", "authors": [ "Dennis Zetner", "Jacob Rosenberg", "Jacob Rosenberg" ], "abstract": "Introduction: Local administration of melatonin might prove useful in future clinical studies. Melatonin possesses poor solubility and stability in aqueous solutions. The aim of this study was to investigate the solubility and stability of melatonin when dissolved in glycofurol, propylene glycol, and dimethyl sulfoxide (DMSO). Methods: Two experiments were performed: solubility and stability. In the solubility experiment, we dissolved melatonin in 20% propylene glycol and 20% glycofurol solutions, respectively. For the stability experiment, we prepared three different formulations: melatonin and glycofurol (20% w/w, 10 mg/g); melatonin, glycofurol, and DMSO (20%, 40% w/w, 10 mg/g); and melatonin and DMSO (50% w/w, 1 mg/g). All three solutions were stored at 25°C for 45 days. Concentrations of melatonin in all solutions were measured through high-performance liquid chromatography. Results: Melatonin demonstrated poor solubility in propylene glycol (3.6–3.8 mg/g) and better solubility in glycofurol (10.5–11.1 mg/g). All three formulations of the stability experiment showed no degradation of melatonin over 45 days. Discussion: Glycofurol and DMSO provide better solubility and stability than aqueous solutions. The formulations used in this experiment have adequate stability to be used in clinical trials.", "keywords": [ "Melatonin", "stability", "solubility", "dimethyl sulfoxide", "DMSO", "glycofurol", "propylene glycol" ], "content": "Introduction\n\nOral melatonin has poor oral bioavailability (DeMuro et al., 2000; Di et al., 1997; Harpsøe et al., 2015; Lane & Moss, 1985). So, if high local doses of melatonin are wanted, other routes of administration might be advantageous, e.g. intravesical, vaginal, rectal, and pulmonal. A liquid solution of melatonin is required for these routes of administration. Since melatonin has poor solubility and stability in aqueous solutions (Hamed et al., 1991), we wanted to investigate alternative solvents. Possible solvents include dimethyl sulfoxide (DMSO), glycofurol, and propylene glycol. DMSO is used as a solvent for intravesical administration of drugs used in the treatment of inflammatory diseases of the bladder (Petrou et al., 2009; Shirley et al., 1978). Glycofurol is considered non-toxic and is used as a solvent in various intravenous formulations (Crowther et al., 1997). Propylene glycol is used extensively in cosmetic products and has been considered safe in this application (Fiume et al., 2012).\n\nThe aim of this study was to investigate the solubility of melatonin in glycofurol and propylene glycol formulations, as well as the stability of melatonin in glycofurol and DMSO formulations.\n\n\nMethods\n\nTwo experiments were performed: a solubility and a stability experiment.\n\nTwo formulations were prepared, one containing 20% w/w glycofurol in type 1 purified (MilliQ) water and the other 20% w/w propylene glycol in purified water. From each formulation, 2 x 1 ml was transferred to separate Eppendorf tubes (1.5 ml). Melatonin was added to each Eppendorf tube in larger quantity than the anticipated aqueous solubility. The Eppendorf tubes were agitated by means of end-over-end rotation overnight. Prior to high-performance liquid chromatography (HPLC) analysis, each sample was filtered (0.45 µm Q-Max RR syringe filters).\n\nFor the stability experiment, the following formulations were prepared:\n\na) 20% w/w glycofurol in MilliQ water containing 10 mg/g melatonin (glycofurol, 1.5 g; MilliQ water, 6 g; melatonin, 75 mg).\n\nb) 20% w/w glycofurol and 40% w/w DMSO in MilliQ water containing 10 mg/g melatonin (Glycofurol, 1.5 g; DMSO, 3 g; MilliQ water, 3 g; melatonin, 75 mg).\n\nc) 50% DMSO in MilliQ water containing 1 mg/g melatonin (DMSO, 3.75 g; MilliQ water, 3.75 g; melatonin, 7.5 mg).\n\nAll formulations were prepared by dissolving the relevant amount of melatonin in the organic solvents. Subsequently, the organic solution was added to the relevant volume of MilliQ water. Each formulation was portioned into 12 Eppendorf tubes. These were stored in a heating cabinet at 25°C for up to 45 days. Three Eppendorf tubes from each formulation were taken for analysis at Day 10, 17, 31 and 45. The amount of melatonin in each formulation was determined immediately after preparation (Day 0). Prior to HPLC analysis, the samples were diluted 40 times in acetonitrile. Settings for the HPLC are listed available as Extended data (Zetner, 2020).\n\n\nResults\n\nThe solubility of melatonin in the prepared 20% w/w propylene glycol and 20% w/w glycofurol formulations is shown in Table 1. The solubility of melatonin in propylene glycol was only 3.6–3.8 mg/ml, while it was 10.5–11.1 mg/ml in glycofurol.\n\nThe results of the melatonin measurements from Day 0 to 45 are shown in Table 2. Melatonin was stable at 25°C for 45 days in all three formulations. None of the melatonin concentrations in the formulations varied considerably from the original concentration. However, when inspecting the HPLC chromatograms of all three products, two peaks were identified in the 20% glycofurol w/w solution at 7.9 and 8.3 minutes. These two peaks were not present in the chromatograms of either solution containing DMSO. All output results are available as Underlying data (Zetner, 2020).\n\nDMSO, Dimethyl sulfoxide, *Only two samples were tested on Day 0.\n\n\nDiscussion\n\nThe solubility of melatonin was nearly three times higher in the glycofurol formulation than in the propylene glycol formulation. A concentration of 10 mg/ml was achieved in the glycofurol formulation. Melatonin concentrations were stable for 45 days in all three formulations of the stability experiment; however, two unidentified peaks were present in the glycofurol solution (Figure 1).\n\nProfiles shown are of melatonin 10 mg/mL in 20% (w/w) glycofurol, 10 mg/mL in 20% w/w glycofurol and 40% w/w dimethyl sulfoxide (DMSO), and 1 mg/g 50% DMSO stored for 45 days at 25°C.\n\nPrevious studies of melatonin stability in aqueous solutions have documented varying results. One study demonstrated stable melatonin concentrations of 100–113 µg/ml in a solution consisting of 5% ethanol and 95% isotonic saline for at least 6 months. The solution was created in sterile conditions and kept in sterile vacuum tubes, protected from light, at room temperature, 4°C, and -70°C (Cavallo & Hassan, 1995). Interestingly, another study investigated the stability of melatonin at 50 µg/ml dissolved in a phosphate buffer at pH 1.2, 2, 4, 7.4, 7, 10, and 12. This showed that up to 30% of the melatonin degraded over 21 days at all pH ranges. These samples were kept at 20°C and 37°C (Daya et al., 2001). This makes it difficult to draw conclusions about whether melatonin is stable in aqueous solutions, but it seems that melatonin dissolved in aqueous solutions is unreliable to use for clinical trials. Furthermore, the concentrations in these studies might be too small to be relevant in a clinical setting compared with the 10 mg/g achieved in the glycofurol formulation in the present study.\n\nTo our knowledge, this is the first trial investigating the solubility and stability of melatonin dissolved in DMSO, glycofurol, and propylene glycol. Study limitations were present since we only had data for 45 days, and solely at 25°C. Also, we did not make a comparison to melatonin in an aqueous solution.\n\nSince our experiments were performed, propylene glycol has received the dubious honor of being named the American Contact Dermatitis Society's ‘Allergen of the Year 2018’ (Jacob et al., 2018). Adding this to the low solubility of melatonin in propylene glycol makes it hard to recommend using propylene glycol as a solvent for melatonin in clinical settings.\n\nBoth formulations containing DMSO demonstrated sufficient stability. The solution containing only glycofurol showed two unidentified peaks in the chromatogram at 45 days. Therefore, it can be speculated that these two peaks represent degradation products of melatonin. However, further studies aimed at identifying these two peaks are needed before they can be named as degradation products of melatonin. Both glycofurol and DMSO provide practical and relatively cheap ways of storing melatonin in a liquid solution. The stability of the DMSO formulations is good enough for them to be used for pharmacokinetic and safety trials in humans. If the formulations are to be used commercially in the long term, a longer stability experiment will have to be performed to determine a clinically relevant shelf life and requirements for storage temperatures.\n\n\nConclusion\n\nThe solubility of melatonin in propylene glycol was low, but melatonin was easily soluble in glycofurol. Glycofurol alone demonstrated sufficient stability, but also showed two unidentified peaks in the chromatogram. Both glycofurol/DMSO, and DMSO alone demonstrated a sufficient stability for melatonin solutions over 45 days at room temperature.\n\n\nData availability\n\nOpen Science Framework: Solubility and stability of melatonin in propylene glycol, glycofurol, and dimethyl sulfoxide. https://doi.org/10.17605/OSF.IO/N9Y7V (Zetner, 2020).\n\nThis project contains the following underlying data:\n\nData.xlsx (All results of HPLC analysis).\n\nChromatograms.xlsx (Chromatograms for Day 0–45 measurements).\n\nOpen Science Framework: Solubility and stability of melatonin in propylene glycol, glycofurol, and dimethyl sulfoxide. https://doi.org/10.17605/OSF.IO/N9Y7V (Zetner, 2020).\n\nThis project contains the following extended data:\n\nAppendix 1 (Settings used for the HPLC-analysis).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nCavallo A, Hassan M: Stability of melatonin in aqueous solution. J Pineal Res. 1995; 18(2): 90–92. PubMed Abstract \| Publisher Full Text\n\nCrowther M, Pilling A, Owen K: The evaluation of glycofurol as a vehicle for use in toxicity studies. Hum Exp Toxicol. 1997; 16(7): 406. Reference Source\n\nDaya S, Walker RB, Glass BD, et al.: The effect of variations in pH and temperature on stability of melatonin in aqueous solution. J Pineal Res. 2001; 31(2): 155–158. PubMed Abstract \| Publisher Full Text\n\nDeMuro RL, Nafziger AN, Blask DE, et al.: The absolute bioavailability of oral melatonin. J Clin Pharmacol. 2000; 40(7): 781–784. PubMed Abstract \| Publisher Full Text\n\nDi WL, Kadva A, Johnston A, et al.: Variable bioavailability of oral melatonin. N Engl J Med. 1997; 336(14): 1028–1029. PubMed Abstract \| Publisher Full Text\n\nFiume MM, Bergfeld WF, Belsito DV, et al.: Safety assessment of propylene glycol, tripropylene glycol, and PPGs as used in cosmetics. Int J Toxicol. 2012; 31(5 Suppl): 245S–60S. PubMed Abstract \| Publisher Full Text\n\nHamed MY, Mostafa EA, Tous SS: Antifertility effect of orally formulated melatonin tablets in mice. Int J Pharm. 1991; 69(2): 93–102. Publisher Full Text\n\nHarpsøe NG, Andersen LP, Gogenur I, et al.: Clinical pharmacokinetics of melatonin: a systematic review. Eur J Clin Pharmacol. 2015; 71(8): 901–909. PubMed Abstract \| Publisher Full Text\n\nJacob SE, Scheman A, McGowan MA: Propylene glycol. Dermatitis. 2018; 29(1): 3–5. PubMed Abstract \| Publisher Full Text\n\nLane EA, Moss HB: Pharmacokinetics of melatonin in man: first pass hepatic metabolism. J Clin Endocrinol Metab. 1985; 61(6): 1214–1216. PubMed Abstract \| Publisher Full Text\n\nPetrou SP, Parker AS, Crook JE, et al.: Botulinum a toxin/dimethyl sulfoxide bladder instillations for women with refractory idiopathic detrusor overactivity: a phase 1/2 study. Mayo Clin Proc. 2009; 84(8): 702–706. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShirley SW, Stewart BH, Mirelman S: Dimethyl sulfoxide in treatment of inflammatory genitourinary disorders. Urology. 1978; 11(3): 215–220. PubMed Abstract \| Publisher Full Text\n\nZetner D: Solubility and stability of melatonin in propylene glycol, glycofurol, and dimethyl sulfoxide. 2020. http://www.doi.org/10.17605/OSF.IO/N9Y7V" }	[ { "id": "80743", "date": "18 Mar 2021", "name": "Marcel Henrique Marcondes Sari", "expertise": [ "Reviewer Expertise Nanotechnology", "pharmaceutical formula development", "biochemistry and toxicology", "pharmaceutical technology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present study provides important data regarding the solubility of melatonin and its stability in different solvents. The document is well-written and well-organized, which facilitates comprehension. During my reading, I identified a few issues that could be considered in the revision process.\nThe title is not informative. It should contain the main findings of the study.\n\nIn the introduction section, the authors could mention the clinical applications of melatonin and reinforce the importance of obtaining a suitable solvent for solubilizing this substance.\n\nMethods – All the materials and reagents used in this study as well as the source of them must be informed.\n\nWater solubility of melatonin for comparative purposes should be included to demonstrate if the proposed solvents improved the stability of the active substance.\n\nMelatonin solubility in DMSO was not provided. Please, include it.\n\nThe authors could attach both chromatograms obtained in the stability evaluation (day 0 and 45) to better demonstrate the profiles. It would improve comprehension and readability.\n\nStatistical evaluation of the data is missing and must be included in order to reinforce the conclusions made. More details regarding data expression and treatment must be informed.\n\nThe figures’ resolution is not optimal. Besides, it is difficult to read the “melatonin” identification at the peak. The authors should improve figures’ quality and also increase the font size.\n\nToxic issues must be considered in selecting an adequate solvent. Besides, the biocompatibility of substances is another critical concern. The authors should include a brief paragraph regarding such subjects in the discussion section.\n\nAny idea regarding the superior stability of melatonin in DMSO solvent? There are some studies that demonstrated the potential antioxidant property of DMSO as a protective effect against melatonin oxidation (DOI 10.1002/nau.232041). The authors could improve the discussion based on such scientific evidence.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate? No\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "88963", "date": "16 Jul 2021", "name": "Aroonsri Priprem", "expertise": [ "Reviewer Expertise pharmaceutical technology", "physicochemical characteristics", "melatonin" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall If the formulations used in this study is to be used in clinical trials, as mentioned, it should provide concrete evidence on safety of the formulation and stability of melatonin (including potential degraded contaminant), as exemplified in a study: http://dx.doi.org/10.1016/j.jpha.2017.04.001.\nSpecific comments\nThe rationale of local administration of melatonin in a solution dosage form and the solvents or solvent mixes which were selected for studies are not fully addressed. Additional citations are essential to support the rationale.\n\nValidations of all tests, particularly HPLC, are not provided. Controls and standards are not adequately provided. Without validated results, controls and standards, it is not certain if the results are reproducible.\n\nThere is no statistical analysis in all tests. Interpretation of stability studies can only be conclusive after statistical analysis of the data.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate? No\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-85
https://f1000research.com/articles/8-1066/v1	11 Jul 19	{ "type": "Research Article", "title": "Pipeline analysis of a vaccine candidate portfolio for diseases of poverty using the Portfolio-To-Impact modelling tool", "authors": [ "Alexander Gunn", "Shashika Bandara", "Gavin Yamey", "Flavia D´Alessio", "Hilde Depraetere", "Sophie Houard", "Nicola K Viebig", "Stefan Jungbluth", "Alexander Gunn", "Shashika Bandara", "Gavin Yamey", "Flavia D´Alessio", "Hilde Depraetere", "Sophie Houard", "Nicola K Viebig" ], "abstract": "Background: The Portfolio-To-Impact (P2I) P2I model is a recently developed product portfolio tool that enables users to estimate the funding needs to move a portfolio of candidate health products, such as vaccines and drugs, along the product development path from late stage preclinical to phase III clinical trials, as well as potential product launches over time. In this study we describe the use of this tool for analysing the vaccine portfolio of the European Vaccine Initiative (EVI). This portfolio includes vaccine candidates for various diseases of poverty and emerging infectious diseases at different stages of development. Methods: Portfolio analyses were conducted using the existing assumptions integrated in the P2I tool, as well as modified assumptions for costs, cycle times, and probabilities of success based on EVI’s own internal data related to vaccine development. Results: According to the P2I tool, the total estimated cost to move the 18 candidates currently in the EVI portfolio along the pipeline to launch would be about US $470 million, and there would be 0.69 cumulative expected launches during the period 2019-2031. Running of the model using EVI-internal parameters resulted in a significant increase in the expected product launches. Conclusions: The P2I tool's underlying assumptions could not be tested in our study due to lack of data available. Nevertheless, we expect that the accelerated clinical testing of vaccines (and drugs) based on the use of controlled human infection models that are increasingly available, as well as the accelerated approval by regulatory authorities that exists for example for serious conditions, will speed up product development and result in significant cost reduction. Project findings as well as potential future modifications of the P2I tool are discussed with the aim to improve the underlying methodology of the P2I model.", "keywords": [ "European Vaccine Initiative", "vaccines", "diseases of poverty", "emerging infectious diseases", "portfolio", "P2I" ], "content": "Introduction\n\nThe Special Programme for Research and Training in Tropical Diseases (TDR) recently developed a new modelling tool, called Portfolio-to-Impact (P2I), that allows users to model the impact of different research portfolios1. The P2I tool can be used to estimate the costs of moving a portfolio of candidate products for poverty-related and neglected diseases (PRNDs) through the pipeline and the likely launches that would result. It can also help to identify potential funding bottlenecks and operational challenges. The modelling tool, which is deterministic, uses Excel based software to estimate the “minimum funding needs to accelerate health product development from late stage preclinical study to phase III clinical trials” and to “visualize potential product launches over time1.”\n\nTo the best of our knowledge, the first published use of the tool was by Young and colleagues2. These researchers first conducted a pipeline portfolio review to identify current candidates in the pipeline for 35 PRNDs. They then used the P2I tool to estimate (a) the costs to move these candidates through the pipeline, (b) the likely launches, and (c) the highly needed products that would still be “missing” at the end. As of August 31, 2017, they found 685 PRND product candidates, of which 538 candidates met inclusion criteria for input into the model. Their modelling estimated that it would cost about US$ 16.3 billion (range $13.4–19.8B) to move these candidates along the pipeline, resulting in about 128 (89–160) expected product launches. The study found that “there would be few launches of complex new chemical entities; launches of highly efficacious HIV, tuberculosis, or malaria vaccines would be unlikely2.”\n\nThe European Vaccine Initiative (EVI), established in 1998 as the European Malaria Vaccine Initiative (EMVI), is a not-for-profit organization that supports the development of effective, affordable and accessible vaccines against diseases of poverty and emerging infectious diseases. To achieve this goal, EVI supports translational vaccine research and development (R&D) spanning from preclinical development through to the establishment of a clinical proof of concept. To date, EVI has supported the development of about 40 vaccine candidates through to early- and mid-stages of clinical development. Initially focussing only on malaria vaccines, in 2009 in the context of a strategic revision EVI broadened its scope and since has built a vaccine portfolio that addresses critical challenges and opportunities in vaccine R&D for a variety of diseases of poverty and emerging infectious diseases.\n\nCurrently EVI´s vaccine portfolio comprises around 20 vaccine candidates at different stages of development between late preclinical and mid-stage clinical development. In order to estimate future financing needs required for delivering the EVI portfolio and the potential public health impact of product launches, we conducted an analysis of EVI´s vaccine candidate portfolio using the P2I tool.\n\nTogether with similar pipeline portfolio reviews using the P2I tool that are currently being conducted by other product development partnerships (PDPs), the results will inform product developers as well as funders and policy makers regarding future funding needs. The results may also guide future investment priorities to maximise the chances of developing products for diseases that are missing urgently needed health products.\n\n\nMethods\n\nIn this section, we describe the four key steps in this analysis, which are summarized in Figure 1. We do not describe the development of the first version of the P2I tool itself (P2I version 1, or P2I v.1), because this has been previously published1. The development of a second version of the P2I tool, called P2I version 2 (P2I v.2), has also been previously described2. In brief, the model is based on assumptions for costs per phase, attrition rates (probability of success) per phase, and cycle times per phase for four development phases (preclinical to phase III) for 11 different kinds of medical products, called archetypes. The P2I tool is a Microsoft Excel based tool and P2I v.2 uses Microsoft Excel 2016.\n\nThe first step was to review, organize, and classify the vaccine candidates to allow them to be entered into the P2I model. To enter candidates into the model, we needed to include (a) the target disease, (b) the current phase of development (the model assumes that the candidate is at the start of that phase), and (c) the archetype. Table 1 summarizes the vaccine candidates included in the analysis. Since all product archetypes were vaccines, we used the archetype classification from the P2I v.2 model (Table 2), in which candidates are classified as simple, complex, or unprecedented.\n\nAbbreviations: *ETEC: enterotoxigenic E. coli\n\nOnce EVI’s portfolio of candidates was classified into archetypes (see Table 1), we then entered them into the P2I v.2 model and ran the model. In this first run of the model, we used exactly the same assumptions on cycle time, cost, and attrition rate per phase as in the P2I.v2 model2. These assumptions are shown in Table 3. The assumptions were derived from three sources: the P2I model (shown in orange in Table 3), the McKinsey Risk-Adjusted Portfolio (RAP) Model (shown in yellow), and the Bill & Melinda Gates Foundation (shown in blue)2. The model outputs were the estimated costs to move the current candidates through the pipeline and the estimated number of launches.\n\nThis table is adapted from reference 2 under a CC-By 4.0 license.\n\nAs a third step, we did a second run of the model after making selected modifications to some of the assumptions used in P2I v.2.\n\nEVI has collected data on its own parameters for cycle times, attrition rates, and costs per phase, which are shown in Table 4. We wanted to assess what effect the use of EVI’s parameters would have upon the outputs of the P2I model, but we recognized that many of these parameters were based on only two or three data points and were thus unreliable. For the second run of the model, we therefore made a pragmatic decision to use only those EVI parameters that were based on 10 data points, i.e. EVI’s parameters on success rate in phase 1 and the duration of phase 1. Thus, in the second run of the model, we made two modifications:\n\nInstead of using a success rate of 50% for phase 1 for unprecedented vaccines (the assumption in P2I v.2, as shown in Table 3), we used EVI’s success rate of 70% (see Table 4).\n\nInstead of using a phase length of 2 years for phase 1 for unprecedented vaccines (the assumption in P2I v.2, as shown in Table 3), we used EVI’s phase length of 1.45 years or 17.4 months (see Table 4).\n\nFor the EVI parameters that were included in the second run of the model, we used the following definitions:\n\nSuccess rate: EVI uses two different measures: (i) technical success: a clinical trial is considered successful if it concluded without being terminated prematurely for whatever reason, and (ii) phase transition success: a clinical trial is considered successful if after completion of the trial a decision is made to move to a subsequent clinical trial (even if no funding is available to do so; successful in this sense means also if you conduct, for example, another phase I clinical trial of the same antigen with a different formulation, dose, or age group).\n\nClinical trial duration: EVI excludes clinical trial preparation, including dossier preparation and waiting for approval, in the duration. The duration is the period between the start and completion times. The start time is the time point when the clinical study opened for recruitment of participants, or the actual date on which the first participant was enrolled. The completion time is the time point when the final participant was examined or received an intervention for the purposes of final collection of data for the primary outcome.\n\nAs a final step, we conducted a sensitivity analysis, using an approach developed by Mestre-Ferrandiz et al. at the United Kingdom Office of Health Economics3. We examined the impact of changing all probabilities of success per phase to 10% higher and 10% lower, and all costs per phase to 10% higher and lower. We also examined the impact of all possible combinations of these changes (e.g., 10% higher probability of success per phase and a 10% higher cost per phase, 10% higher probability of success per phase and a 10% lower cost per phase, etc.). We conducted this sensitivity analysis for both runs of the model.\n\n\nResults\n\nA review of EVI’s portfolio identified a total of 18 vaccine candidates under development, which were entered into the P2I v.2 model (Table 1). There were 8 candidates for malaria, 5 for placental malaria, 2 for leishmaniasis, and one each for shigellosis, Nipah virus, and Zika virus. Three candidates were in the pre-clinical phase, 11 in phase I, and 4 in phase II. There were no candidates in phase III. Among the 18 candidates, 15 were classified as unprecedented vaccines, 2 as simple vaccines, and one as a complex vaccine.\n\nAs described in the Methods section above, we first ran the model using the assumptions included in the P2I v.2 model. The results of this first run are described in section (ii) below. We then re-ran the model with some modifications to the assumptions that were made by EVI, based on EVI’s own historical data; section (iii) describes the results of this second run. Finally, we conducted a sensitivity analysis around the results of both runs of the model; the results of these sensitivity analyses are in section (iv).\n\nEstimated costs to move candidates through the pipeline. The total estimated cost to move the 18 candidates along the pipeline to launch would be about US $470 million (Table 5, Figure 2). Just over one third (35%) of the expected costs would be incurred by development of the 8 vaccine candidates for malaria. Development of candidates for placental malaria, Nipah virus, and Zika virus would each account for about 16% of the total costs. The remaining costs would be for development of candidates for leishmaniasis (10% of costs) and shigellosis (7% of costs).\n\nExpected launches. Overall, for all 18 candidates under development, the P2I model estimates that there would be 0.69 cumulative expected launches (we have left all results unrounded). Figure 3 summarizes the expected launches by disease. Table 6 summarizes the estimated cumulative launches by year from 2019-2031 alongside cost estimates. Figure 4 shows the time trends in cumulative costs and cumulative launch probability from 2019-31.\n\nThe re-run (second run) of the model using the modified assumptions increased the projected portfolio costs by US $46 million, bringing the total cost estimate to US$ 517 million to move all 18 candidates through the pipeline. The changes in estimated cost are driven by the increase in expected costs for the unprecedented vaccine candidates, i.e., the 8 malaria vaccine candidates, 5 placental malaria vaccine candidates and 2 leishmaniasis vaccine candidates. The remaining candidates for Nipah virus, Zika virus, and shigellosis were not affected by the change in parameters.\n\nWith regards to the expected number of launches, in the re-run of the model, the launch probability increased for malaria, placental malaria, and leishmaniasis vaccines:\n\nFor malaria, the estimate of expected launches increased from 0.098 to 0.11\n\nFor placental malaria, the estimate of expected launches increased from 0.05 to 0.07\n\nFor leishmaniasis, the estimate of expected launches increased from 0.024 to 0.03.\n\nTable 7 provides a comparative summary of the costs and launch probabilities for each run of the model.\n\nWe conducted a sensitivity analysis for the first and second runs of the model.\n\nSensitivity analysis for first run of the model. In the sensitivity analysis for the first run of the model (which used assumptions from P2I v.2), we found that the total estimated costs ranged from US$ 482.48 million to US$ 581.9 million (Table 8). The launch probability ranged from 0.53 to 0.96.\n\nSensitivity analysis for second run of the model. In the second sensitivity analysis for the second run of the model (which used modified assumptions), we found that the total estimated cost ranged from US $417.08 million to US $528.11 million (Table 9). The launch probability ranged from 0.51 to 0.91.\n\n\nConclusions and discussion\n\nThe mission of EVI is to accelerate the development of vaccines for diseases of poverty and emerging infectious diseases. Compared to other PDPs with a narrower focus, for example on a single disease, EVI has a broader scope and consequently a more heterogeneous portfolio, currently comprising 18 active vaccine candidates covering five different diseases/pathogens (malaria, including placental malaria, leishmaniasis, shigella/ETEC, Nipah and Zika viruses). An analysis of EVI´s current vaccine portfolio—providing estimations for future vaccine development costs and expected product launches—was considered important to inform EVI´s decision-making and priority setting, as well as to provide valuable information to global health funders and policy makers.\n\nOverall, using the pre-defined assumptions established in the P2I tool, our modelling resulted in a total estimated cost of about US $470 million for moving all 18 candidates included in the analysis along the pipeline until launch. Of this total amount, just over one third (35%) of the expected costs would be incurred by the development of the eight vaccine candidates for malaria (excluding those for placental malaria). Development of the candidates for placental malaria, Nipah virus, and Zika virus would account for about 16% each of the total costs. The remaining financing would be required for the development of candidates for leishmaniasis and shigellosis (10% and 7% of total costs, respectively).\n\nThe re-run (second run) of the P2I model using modified assumptions for phase costs and length based on EVI’s internal data increased the projected portfolio costs by US $46 million up to a total cost of US$ 517 million for all 18 candidates. The main driver of this change in the estimated cost is the increase in expected costs for the unprecedented vaccine candidates (i.e. the eight malaria vaccine candidates, five placental malaria vaccine candidates and two leishmaniasis vaccine candidates). Results for the vaccine candidates for Nipah virus, Zika virus, and shigellosis, ETEC were not affected by the change in these parameters.\n\nThe costs that we estimated using the P2I tool are likely to be an underestimate of the true costs. Vaccine development is a reiterative process, meaning that many steps, such as clinical trial phases, will be conducted several times. This process is in contrast to the rationale of the P2I tool, which assumes a straightforward development of product candidates without the reiteration of any particular development steps. For example, very often several phase I trials for a particular antigen are conducted, i.e. multiple trials in which different formulations, different routes of administration or different technology platforms for antigen presentation are being tested and compared. Also, very often several phase I clinical trials are conducted in which a vaccine´s safety is assessed in various age groups in an age-deescalating manner (i.e. the vaccine is tested consecutively in volunteer groups with decreasing age). It is therefore rather unlikely that any vaccine candidate would immediately advance to a phase II clinical trial after the conduct of a single phase I clinical trial only. Consequently, the total costs for the development of the different vaccine candidates in EVI´s portfolio are likely to be significantly higher than those estimated by the P2I tool. In addition, as several phase I or phase II clinical trials are likely to be conducted for the same vaccine (as explained above), due to such “reiterated” phases the overall timelines to reach the market are expected to be longer.\n\nWith regards to the estimated future launches, for all 18 candidates under development, the P2I model estimates that there would be 0.69 cumulative expected launches. In the re-run of the model using EVI‘s own internal parameters, thanks to higher success rates at EVI as compared to the P2I tool´s predefined parameters, the launch probability increased for malaria, placental malaria, and leishmaniasis vaccines (from 0.098 to 0.11, from 0.05 to 0.07, and from 0.024 to 0.03, respectively). Directly related to this increased launch probability, the total estimated costs for moving these vaccine candidates through the pipeline increased accordingly. This difference between the estimated future product launches emphasizes that, in order to increase the chances of ultimate success with this kind of product development, it is important to make the product development process technically as efficient as possible, i.e. reducing the attrition rates as much as possible. If attrition rates during product development cannot be reduced, the only other chance of achieving ultimate product launches is to boost the overall number of product candidates in the pipeline, obviously in the end resulting in higher total costs linked to the launch of a single product due to the high costs linked to failed product candidates.\n\nHowever, rather than looking at the isolated results on likely launches from the analysis of a single organization´s portfolio, a more meaningful estimation will be obtained by conducting a much wider portfolio analysis in which the launch estimation results of the entire global vaccine candidate portfolio are estimated in an integrated, combined manner. Only this kind of “full portfolio” study can provide a reliable prediction of the product success rates on a global level for the next few decades.\n\nThe P2I tool has a number of other limitations, which were described in detail by Young et al.2 These include: (i) as a static, deterministic model, it does not take into account possible improvements in product development techniques over time (e.g. R&D efficiencies that reduce costs); (ii) the model’s assumptions for costs, attrition rates, and cycle times for phase are based on product development data from across multiple diseases (including non-communicable diseases—the model does not reflect possible differences in R&D parameters between different diseases); and (iii) the model does not include all phases of development (e.g. it excludes drug discovery, basic research, and regulatory review).\n\nIn this study, several adaptations to the P2I tool initially were considered with the aim of improving the tool´s usefulness and reliability. First, we considered making adaptations of the assumptions for success rates, costs and cycle times based on EVI’s longstanding experience with conducting studies in resource-limited, low-income settings. Second, we considered making adaptations for the same parameters based on (a) the fast-track clinical development strategy often used by EVI (consisting of a strategy in which the first-in-human evaluation involves a staggered multi-centre phase Ia/b clinical trial resulting in shorter timelines4,5), and (b) the inclusion of accelerated clinical testing based on controlled human infection models available, for example, for malaria6,7. Third, in the original proposal we considered including adaptations of the assumptions for costs and cycle times for vaccines that might be eligible for accelerated approval by regulatory authorities, for example the “Expedited Programs for Serious Conditions––Drugs and Biologics” from the US Food and Drug Administration8.\n\nIn the end, we were able to do one re-run of the P2I model using EVI’s own parameters for success rates and cycle times for phase I clinical trials for unprecedented vaccines (70% and 17.4 months, respectively, compared to 50% and 24 months defined in the original P2I tool for unprecedented vaccines). However, although to date EVI has been involved in the conduct of over 30 clinical trials, data from only a limited number of studies could be used in the analyses conducted. When it came to estimating clinical trial costs, for example, for several trials it was not possible to extract the specific costs for preclinical or clinical trial activities out of the overall study costs.\n\nConcerning the proposed modification of P2I parameters based on accelerated clinical testing using controlled human challenge models and on accelerated approval by regulators, we realized that although these two issues are likely to speed up vaccine development, currently there is not enough evidence/data based on which the P2I parameters could be adapted and analyses be run to assess their impact.\n\nIn conclusion, we found that despite the limitations discussed above, the P2I tool was flexible and adaptable enough to be used to study EVI’s portfolio. We believe that the P2I model represents a useful tool to analyze the portfolio of global health products under development. We expect that studies like ours will inform future updates of the model that will further increase its value for product developers, R&D funders, and decision makers.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nThe particular vaccine candidates included in this study have been anonymized for intellectual property reasons.", "appendix": "Grant information\n\nThis work, Project ID N⍜ B80106, received financial support from TDR, the Special Programme for Research and Training in Tropical Diseases, co-sponsored by UNICEF, UNDP, the World Bank and WHO.\n\nThe funders had no role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe would like to thank Robert Terry at TDR for his guidance on this study.\n\n\nReferences\n\nTerry RF, Yamey G, Miyazaki-Krause R, et al.: Funding global health product R&D: The Portfolio-To-Impact Model (P2I), a new tool for modelling the impact of different research portfolios [version 2; peer review: 2 approved]. Gates Open Res. 2018; 2: 24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoung R, Bekele T, Gunn A, et al.: Developing new health technologies for neglected diseases: a pipeline portfolio review and cost model [version 1; peer review: 1 approved, 2 approved with reservations]. Gates Open Res. 2018; 2: 23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMestre-Ferrandiz J, Sussex J, Towse A: The R&D Cost of a New Medicine. UK Office of Health Economics. 2012. Reference Source\n\nSteiner-Monard V, Kamaka K, Karoui O, et al.: The Candidate Blood-stage Malaria Vaccine P27A Induces a Robust Humoral Response in a Fast Track to the Field Phase 1 Trial in Exposed and Nonexposed Volunteers. Clin Infect Dis. 2019; 68(3): 466–474. PubMed Abstract \| Publisher Full Text\n\nSirima SB, Durier C, Kara L, et al.: Safety and immunogenicity of a recombinant Plasmodium falciparum AMA1-DiCo malaria vaccine adjuvanted with GLA-SE or Alhydrogel® in European and African adults: A phase 1a/1b, randomized, double-blind multi-centre trial. Vaccine. 2017; 35(45): 6218–6227. PubMed Abstract \| Publisher Full Text\n\nRoestenberg M, Mordmüller B, Ockenhouse C, et al.: The frontline of controlled human malaria infections: A report from the controlled human infection models Workshop in Leiden University Medical Centre 5 May 2016. Vaccine. 2017; 35(51): 7065–7069. PubMed Abstract \| Publisher Full Text\n\nHodgson SH, Juma E, Salim A, et al.: Lessons learnt from the first controlled human malaria infection study conducted in Nairobi, Kenya. Malar J. 2015; 14: 182. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nExpedited Programs for Serious Conditions - Drugs and Biologics. US Food and Drug Administration, 2017. Reference Source" }	[ { "id": "52206", "date": "13 Aug 2019", "name": "Jean-Louis Excler", "expertise": [ "Reviewer Expertise Vaccine Development", "Global Health" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors use the Portfolio-To-Impact (P2I) version model to estimate funding needs to move candidate vaccines to potential launch. P2I is a cost modeling tool.\n\nIt is unclear to a reader unfamiliar with this tool who are the target stakeholders of the outcome of the analysis: manufacturers, non-for-profit developers, donors, public health policy makers? Although the authors provide references, this should be summarized and clarified.\n\nIt is also unclear how this tool articulates for a given vaccine with the development of the Business Case component of the Full Public Health Value Proposition and the Total Systems Effectiveness (TSE) and the Vaccine Innovation Prioritization Strategy (VIPS) developed by WHO. What is the added value of the P2I model compared to the other models referred in Table 3? This should also be clarified.\n\nIt might also be useful to remind how EVI prioritized their vaccine candidate portfolio (no reference cited) and why they selected some of them for this P2I analysis.\n\nThe clinical trial duration as defined on page 6 may be misleading. The added duration of safety and immunogenicity analysis (the latter may be long) is not taken into account while these are key data driving the next phases of development.\nThe term \"launch\" may deserve some clarification. Are the authors talking about registration for launch on the public market in LMICs, private market in LMICs, or per Gavi scheme for eligible countries? What about MICs which are no longer Gave-eligible and whose list is increasing? It is unclear whether the model considers vaccine manufacturing by traditional big western pharmas or developing country vaccine manufacturers (DCVM) and consider bringing the vaccine to WHO prequalification. This latter point is of importance as often this may require additional clinical studies adding to the overall development costs and duration before launch.\n\nIt is difficult to understand Tables 6-7 (Title of Table 7: delete 'of' based...) and Figures 3-4 of 'expected launches'. What do the data presented mean? For example, in text '0.69'. Is it a probability? Similarly Tables 8 and 9 would need ample explanation about what is considered in these tables.\n\nComing back to the estimated cost of vaccine development to launch, it is difficult to weigh the value of the findings compared to real life costs. For example, the cost of RTS,S malaria vaccine developed by GSK is estimated to be close to $500M. For Nipah and Zika, what is the development cost estimated by CEPI? How does it compare to EVI estimates? What the P2I model ever tested a posteriori for vaccines already developed and for which the development cost is know in order to measure the accuracy of the model prediction?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5137", "date": "04 Feb 2020", "name": "Stefan Jungbluth", "role": "Author Response", "response": "Comment: It is unclear to a reader unfamiliar with this tool who are the target stakeholders of the outcome of the analysis: manufacturers, non-for-profit developers, donors, public health policy makers? Although the authors provide references, this should be summarized and clarified Response from authors: We have added a sentence in the introduction to explain this. We now state: “As a financial forecasting tool that estimates the funding needs for pharmaceutical product development, the tool and its outputs are of value to funders of product development, product development partnerships, and other stakeholders involved in research and development (R&D) policy. Terry and colleagues, the developers of the P2I model, note that “its real utility lies in its predictive value for modelling the impact of different funding strategies at the portfolio level.”1 Comment: It is also unclear how this tool articulates for a given vaccine with the development of the Business Case component of the Full Public Health Value Proposition and the Total Systems Effectiveness (TSE) and the Vaccine Innovation Prioritization Strategy (VIPS) developed by WHO. Response from authors: In the introduction, we have now added: “We used the P2I tool because, to the best of our knowledge, it is the first publicly available comprehensive portfolio model that includes data on cost, success rate, and cycle time per phase for various product types based on data from a very large number of previous product development candidates (over 25,000)1. The P2I tool is thus complementary to other available tools that can help guide prioritization in vaccine development, such as the multi-stakeholder Vaccine Innovation Prioritization Strategy and Total Systems Effectiveness Framework.” Comment: What is the added value of the P2I model compared to the other models referred in Table 3? This should also be clarified. Response from authors: Table 3 does not show other models. It shows the P2I model’s underlying assumptions on cost, cycle time, and success rate per development phase for three different categories of vaccines (simple, complex, and unprecedented). It also shows the source of data for these assumptions. In the text, we state: “In this first run of the model, we used exactly the same assumptions on cycle time, cost, and attrition rate per phase as in the P2I.v2 model 2 . These assumptions are shown in Table 3. The assumptions were derived from three sources: the P2I model (shown in orange in Table 3), the McKinsey Risk-Adjusted Portfolio (RAP) Model (shown in yellow), and the Bill & Melinda Gates Foundation (shown in blue) 2 .” Comment: It might also be useful to remind how EVI prioritized their vaccine candidate portfolio (no reference cited) and why they selected some of them for this P2I analysis. Response from authors: New wording describing the vaccine candidate selection and priorisation process, including a reference, have been included in the discussion. Reasons re the selection of vaccine candidates that were included in this study were already provided in the original article. Comment: The clinical trial duration as defined on page 6 may be misleading. The added duration of safety and immunogenicity analysis (the latter may be long) is not taken into account while these are key data driving the next phases of development. Response from authors: This is a valid point. The model only includes advanced clinical to phase III (it does not include phase IV (examining for safety and side effects). We now discuss this limitation in greater detail in the discussion. In the discussion section, we have added: “The exclusion of phase IV studies, also known as post-marketing surveillance, is a major limitation—determining long-term safety and effectiveness is critical, yet it can be a lengthy, expensive process.” Comment: The term \"launch\" may deserve some clarification. Are the authors talking about registration for launch on the public market in LMICs, private market in LMICs, or per Gavi scheme for eligible countries? What about MICs which are no longer Gavi-eligible and whose list is increasing? It is unclear whether the model considers vaccine manufacturing by traditional big western pharmas or developing country vaccine manufacturers (DCVM) and consider bringing the vaccine to WHO prequalification. This latter point is of importance as often this may require additional clinical studies adding to the overall development costs and duration before launch. Response from authors: This is an excellent point (reviewer 2 also raised this point); we have now clarified this in more detail. Several reviewers asked for more details about what was included and what was excluded from the P2I model. This level of detail is in the original paper that describes the P2I model development (https://gatesopenresearch.org/articles/2-24/v2), but since the reviewers has asked to see the details, we have now included these in our revised paper. We use the word “launch” here to refer to a candidate making it through Phase III, i.e., it is “launch ready,” which we now state in the revised paper. We have added a figure to show which phases are included in the P2I model. The P2I model excludes all costs related to basic research through lead optimization; chemistry, manufacturing, and controls; good manufacturing practice; manufacturing build up and scale-up costs; regulatory or registration fees (post-phase III); and all post-market commitments (e.g., phase IV pharmacovigilance studies). Comment: It is difficult to understand Tables 6-7 (Title of Table 7: delete 'of' based...) and Figures 3-4 of 'expected launches'. What do the data presented mean? For example, in text '0.69'. Is it a probability? Similarly Tables 8 and 9 would need ample explanation about what is considered in these tables. Response from authors: We have fixed the typo, we have clarified that these are indeed launch probabilities, and we have now added much greater explanation of the findings shown in Tables 6-9 and figures 3-4. Comment: For Nipah and Zika, what is the development cost estimated by CEPI? How does it compare to EVI estimates? Response from authors: As far as we know, no estimated costs for the total or different steps are available from CEPI for the development of Nipah and Zika vaccines. Also, such costs would vary significantly, depending on the particular vaccine technology employed for vaccine development (eg protein-based vs. DNA/RNA based vs. viral vector etc), as well as on many other factors. Data one can obtain from the CEPI web page are the total maximum funding amounts awarded by CEPI for the different Nipah and Zika vaccines that currently are in the CEPI portfolio. However, we do not consider these figures as meaningful comparators for our estimations as the details of the funded projects (eg vaccine technology, other specific activities included in different projects etc) are not known to us and it is therefore unclear how the vaccines funded by CEPI compare to the one in EVI´s portfolio (NB: The Nipah vaccine candidate in EVI´s portfolio is funded by CEPI but vaccine development is only funded down to phase II clinical trial). Comment: What the P2I model ever tested a posteriori for vaccines already developed and for which the development cost is known in order to measure the accuracy of the model prediction? Response from authors: No vaccines that have finalised their development are available at EVI for such an a posteriori analysis, so for the time being this issue cannot be addressed." } ] }, { "id": "51927", "date": "19 Aug 2019", "name": "Gerald H. Voss", "expertise": [ "Reviewer Expertise Vaccine development", "PRDs", "global health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article describes the application of the P2I model to the vaccine development portfolio of EVI. The intent of the exercise is to provide a portfolio funding estimate from late preclinical through completion of phase III. The authors proceed in four steps by classifying their vaccine candidates into archetypes, running the standard P2I v2 model, re-running the model using modified assumptions based on EVI's own data, and conducting a sensitivity analysis for the model assumptions. The authors conclude that the P2I model, despite several limitations, is suitable and sufficiently flexible to study EVI's vaccine portfolio.\nThere are a number of methodological questions that merit further consideration or clarification.\n\nFirst, the notion of product launch is vague. The model calculates costs from late preclinical up to completion of phase III, so this approach seems to provide numbers for 'launch readiness' rather than product launch and should be clearly stated.\nSecond, significant other costs for vaccine development do not seem to be included, for example process development, GMP lots and investment into manufacturing. Due to this limitation, the calculated funding needs provide only a partial view of the overall required investment.\nThird, clinical phase duration does not take into account the time to prepare for clinical trials, perform data analysis and generate a study report. Together with the author's comment that each clinical trial phase is likely to comprise several iterative studies, the time (and cost) assumptions appear very optimistic.\nFourth, the classification into archetypes is somewhat arbitrary. One could argue that the RTS,S and M72 (out of scope of this exercise) vaccines provide precedents for malaria and TB, respectively, and should increase the phase II and III probability of success assumptions.\nOverall, this study seems to demonstrate that the usefulness of any model depends on the quality of the assumptions. Unfortunately, as the authors indicate, there is a scarcity of data in type II and III disease vaccine development, thereby rendering the generation of assumptions unreliable.\nIn the reviewer's opinion, the article would benefit from answering two questions: how has the model helped EVI to prioritise its portfolio and how can the model be improved.\nMinor comments:\nTable 5 and figures 2 and 3 are redundant Table 6 and figure 4 are redundant. It would also help to clarify what 'cost' and 'cumulative annual' means. Is that 'cumulative cost'? Table 7 'of' needs to be deleted in the title\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5138", "date": "04 Feb 2020", "name": "Stefan Jungbluth", "role": "Author Response", "response": "Comment: First, the notion of product launch is vague. The model calculates costs from late preclinical up to completion of phase III, so this approach seems to provide numbers for 'launch readiness' rather than product launch and should be clearly stated Response from authors: Reviewer 1 also raised this point. We now explain this more clearly. We state: “in this paper, the term launch refers to a candidate making it through phase III and thus being ready for the next steps, e.g. regulatory and manufacturing steps.” Comment: Second, significant other costs for vaccine development do not seem to be included, for example process development, GMP lots and investment into manufacturing. Due to this limitation, the calculated funding needs provide only a partial view of the overall required investment. Response from authors: The reviewer is correct, and this is one of the limitations of the P2I model. We now state this limitation clearly in the methods, and discuss its implications in the Discussion section. In the revised Methods, we now state: “The model includes only advanced preclinical to phase III, and thus the cost estimates are an under-estimate of the full costs of product development. In particular, the model excludes all costs related to basic research through lead optimization; chemistry, manufacturing, and controls; good manufacturing practice; manufacturing build up and scale-up costs; regulatory or registration fees (post-phase III); and all post-market commitments (e.g., phase IV pharmacovigilance studies).” Comment: Third, clinical phase duration does not take into account the time to prepare for clinical trials, perform data analysis and generate a study report. Together with the author's comment that each clinical trial phase is likely to comprise several iterative studies, the time (and cost) assumptions appear very optimistic. Response from authors: In the revised paper, we now explain exactly how these assumptions were generated and validated (and also point readers to the two previous research articles that explain these assumptions in greater detail). Comment: Fourth, the classification into archetypes is somewhat arbitrary. One could argue that the RTS,S and M72 (out of scope of this exercise) vaccines provide precedents for malaria and TB, respectively, and should increase the phase II and III probability of success assumptions. Response from authors: We respectfully disagree that classification is arbitrary, and we now refer readers to our previous two studies showing more details on the classification process. But we certainly do agree that classification is an imperfect science, and we now discuss this limitation in the revised Discussion section. Comment: Overall, this study seems to demonstrate that the usefulness of any model depends on the quality of the assumptions. Unfortunately, as the authors indicate, there is a scarcity of data in type II and III disease vaccine development, thereby rendering the generation of assumptions unreliable. Response from authors: We agree that all models are dependent on the quality of the model assumptions. We now explain in greater detail how these were derived and validated. We respectfully disagree that the assumptions are unreliable; indeed, we know of no author assumptions that are more reliable than those in the P2I model. We now state, in the revision: “As described in detail in reference 1, assumptions on development costs at each phase of product development for the 11 archetypes included in the P2I.v1 model were initially based on an analysis of clinical trial costs from Parexel’s R&D cost sourcebook. The assumptions on attrition rates and cycle times at each phase were initially based on the historical attrition rates and cycle times of more than 25,000 development candidates. All of these assumptions were further refined and validated based on academic literature, industry publications and databases, and 133 stakeholder interviews with a wide variety of product development partnerships (PDPs), pharmaceutical companies, and major funders of global health R&D. As described in detail in reference 2, additional sources of assumptions for the new archetypes in P2I.v2 were derived from the McKinsey Risk-Adjusted Portfolio (RAP) Model and from clinical trial data shared with us by the Bill & Melinda Gates Foundation.” In the discussion section, when we discuss limitations, in the revision we now write: “the model’s assumptions for costs, attrition rates, and cycle times for phase are based on product development data from across multiple diseases (including non-communicable diseases—the model does not reflect possible differences in R&D parameters between different diseases). Although the assumptions were based on a very large number of data points (from 25,000 development candidates) and validated with experts, it is unclear how many of these data points came from vaccine development for neglected and emerging infectious diseases. Thus there is some uncertainty as to how accurate the assumptions are for the costs, attrition rates, and cycle times per phase for the three vaccine archetypes used in our study.” Comment: How has the model helped EVI to prioritise its portfolio? Response from authors: Corresponding wording has been included in the article. At this particular moment, the analysis with the P2I tool is too recent and has not yet been taken into account in any particular decision making concerning the vaccine portfolio. Comment: How can the model be improved? Response authors: The Discussion part of the article already contained a lengthy section where weaknesses and potential improvements to the tool are being discussed Other minor suggestions (spelling etc.) made by the reviewer have been included in the revised article. At the reviewer’s suggestion, we have removed figures 2-4. We have also taken out the word “cumulative” throughout the paper, as it was confusing." } ] }, { "id": "53116", "date": "30 Sep 2019", "name": "Maria Elena Bottazzi", "expertise": [ "Reviewer Expertise Neglected tropical diseases", "vaccine development", "product development", "global health" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript attempts to describe the applicability and adaptability (with assumption modifications) of a recently developed computer (Excel)-based modelling tool called Portfolio-to-Impact (P21 - version 2) that has as objective to estimate the minimum funding needs (costs) to move a portfolio of target candidates (drugs or vaccines) from late stage preclinical up to phase 3 trials and estimate the number of targets with the potential to reach the launch stage. Of note, the authors mention that prior publications describe the P21 tools in detail for both Versions 1 and 2 and that a prior publication had already analyzed the application of the P21 tool using a broad portfolio of 538 PRND candidates.\n\nFor this manuscript the authors use as a case study a selection of targets from the vaccine portfolio of the European Vaccine Initiative (EVI). They focus on 18 vaccine candidates (3 in preclinical, 11 in phase 1 and 4 in phase 2) and with three different categories: 15 as unprecedented vaccines (13 malaria and 2 leishmania), 2 as simple vaccines (Zika and nipah) and 1 as a complex vaccine (shigella/ETEC). As methodology, the authors compare the outputs obtained after using the original P21V2 tool assumptions (cycle time, cost and attrition rate) with a modified set of EVI assumptions.\n\nAs a reviewer, I believe the paper has some potential usability and interest, especially for other PRND vaccine developers and PDPs and those with small portfolios. There are several major observations, however that require attention and/or clarification to strengthen the paper before it is to be indexed:\nEven though the paper highlights that the tool was developed specifically for PRNDs, it lacks more detailed information, discussion and evaluation of the bottlenecks or considerations especially when the development is being done outside HICs and the likely the challenges that would be applicable when the vaccines could be developed partly in and with LMICs organizations. The examples using the EVI portfolio still leaves unclear where the different phases are done. What if preclinical is in Europe but the clinical in Africa, would it be different the cost or probability?\n\nThe model and paper don’t seem to take into consideration nor discuss the variable and unpredictable costs during vaccine development. For example, cost of goods, manufacture, proprietary components such as adjuvants, stability, regulatory, quality control costs, all needed to maintain and even replenish the clinical lots during the transition into the advanced clinical stages.\n\nIt also makes no mention on the complexity of the costs, time and probability needed to continuously mature the production processes and its QC testing and reach suitability for use throughout the different clinical stages and pre-launch or post-launch. For instance, when measuring the probability and time to launch, does this take into consideration where and who would be the large-scale manufacturer? Would there also be the need for manufacturing infrastructure?\n\nEven though the manuscript speaks about using “time to launch” and “probability to launch”, this is very vague and offers no value to the reader since there is a big valley after phase 3 trials that is not discussed. The public health value proposition, business case and demand forecasting for PRNDs is a very complex system that require the involvement of multiple WHO offices and committees, GAVI and others. This should be better elaborate in the manuscript.\n\nThe manuscript lacks to elaborate on the reality that even once a product enters into Phase 1 there are bottlenecks, which may require that a product returns to a preclinical evaluation stage, for example if different formulations need to be re-evaluated or alternate adjuvants need to be tested. It also is not clear about costs and time when the clinical development is in resource-poor areas where regulatory hurdles may hamper the timelines.\n\nStep 2 method description and table 3: if in the first run, the authors are using “exactly the same assumptions as P21V2 model”, it is unclear why there is also reference to two additional set of assumptions: the RAP and the BMGF assumptions. Are these readily available? If not, this would make the applicability of P21V2 obsolete especially for other PDPs and PRND vaccine developers with smaller or non-gates supported portfolios.\n\nStep 3 method description and table 4: the authors mention that the modified EVI assumptions may be unreliable because they are based only on 2-3 data points and decided to make a pragmatic decision to use only those with 10 data points. This in itself is flawed. If the objective of the authors is to showcase how the tool assumptions or a modification of tool assumptions could support vaccine producers in their forecasting exercise, the model should be based on close to reality, especially since most PRND vaccine portfolios do not contain large number of vaccine candidates or data points. The authors should address and comment on this especially since it seems the probability rates are too generous in the EVI assumptions.\n\nThe disease focus of the selected portfolio is quite varied and for some (ie malaria) with multiple targets versus for some only one target. A more detail discussion on the applicability of the tool for the evaluation of 1-2 targets versus >2 targets would be very useful.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5139", "date": "04 Feb 2020", "name": "Stefan Jungbluth", "role": "Author Response", "response": "Comment: Even though the paper highlights that the tool was developed specifically for PRNDs, it lacks more detailed information, discussion and evaluation of the bottlenecks or considerations especially when the development is being done outside HICs and the likely the challenges that would be applicable when the vaccines could be developed partly in and with LMICs organizations. The examples using the EVI portfolio still leaves unclear where the different phases are done. What if preclinical is in Europe but the clinical in Africa, would it be different the cost or probability? Response from authors: This is an excellent point. The model is “agnostic” as to where the development is being done, which is a limitation. In the revision, we now acknowledge this limitation in the Discussion section (in the discussion of limitations). We write: “Fifth, the model assumes that the costs, attrition rates, and cycle times per phase for vaccine development would be the same regardless of the setting where the study is done. Yet in reality, it is likely that these parameters would be different if a study was conducted in a high-, middle-, or low-income country. It would be helpful for future iterations of the P2I tool to incorporate these differences across study settings.” Comment: The model and paper don’t seem to take into consideration nor discuss the variable and unpredictable costs during vaccine development. For example, cost of goods, manufacture, proprietary components such as adjuvants, stability, regulatory, quality control costs, all needed to maintain and even replenish the clinical lots during the transition into the advanced clinical stages. It also makes no mention on the complexity of the costs, time and probability needed to continuously mature the production processes and its QC testing and reach suitability for use throughout the different clinical stages and pre-launch or post-launch. For instance, when measuring the probability and time to launch, does this take into consideration where and who would be the large-scale manufacturer? Would there also be the need for manufacturing infrastructure? Response from authors: This is another excellent point. We now discuss this in the limitations section of the discussion. We write: “Sixth, the model also assumes that the costs of vaccine development per phase do not vary and are predictable. Yet there can be substantial variation and unpredictability in items such as the cost of manufacturing the product candidates and adjuvants, or the maintenance and quality control of clinical trial sites.” As discussed in detail in the revision, there are many costs that are excluded from the P2I model, which is indeed a limitation. The model only includes advanced clinical to phase III (it does not include post-phase III large-scale manufacturing, regulatory approval, marketing, etc.). We state: “Third, the model does not include all phases of development (e.g. it excludes discovery, basic research, and regulatory review). The exclusion of phase IV studies, also known as post-marketing surveillance, is a major limitation—determining long-term safety and effectiveness is critical, yet it can be a lengthy, expensive process. We acknowledge that using the P2I tool, which only includes advanced pre-clinical to phase III, will always lead to an under-estimate of the total costs, since the costs of early pre-clinical research and post-phase III research can both be substantial. For example, based on data from a sample of 106 NCEs, DiMasi et al estimate that developing an NCE to the point of marketing approval costs $2.6 billion to, which includes $1.2 billion in “time costs” (the expected returns that private investors forgo while a drug is in development).10 Of this $2.6 billion, $1.1 billion is in pre-clinical development costs and $1.5 billion in clinical development costs. Previous research has suggested that the cost of regulatory approval stage may represent up to 5.7% of the total R&D cost.11” Comment: Even though the manuscript speaks about using “time to launch” and “probability to launch”, this is very vague and offers no value to the reader since there is a big valley after phase 3 trials that is not discussed. The public health value proposition, business case and demand forecasting for PRNDs is a very complex system that require the involvement of multiple WHO offices and committees, GAVI and others. This should be better elaborate in the manuscript. Response from authors: In the revision, we now make it much clearer what we mean by launch (i.e. a candidate makes it through phase III and is ready for post-phase III steps, such as regulatory review). We also now, in the discussion, explore the valley after phase III. In the revision, we now state: “By only including advanced pre-clinical to phase III, the model also provides no insight into the costs and complexity of the array of activities that need to happen after phase III for a new product to have a public health impact. The phase after phase III is often considered as a “valley of death” for product development—a product may pass successfully through phase III but then there is no concerted, strategic plan for large-scale manufacturing or scale-up. Demand forecasting, developing a long-term business case, understanding the public health value of new products, and analysing the delivery system and scale-up approach are all critical components in determining the ultimate public health utility of a new health technology. We have previously noted that the P2I model “is “agnostic” when it comes to the public health value of the estimated launches—it cannot judge their clinical utility.”2” Comment: The manuscript lacks to elaborate on the reality that even once a product enters into Phase 1 there are bottlenecks, which may require that a product returns to a preclinical evaluation stage, for example if different formulations need to be re-evaluated or alternate adjuvants need to be tested. It also is not clear about costs and time when the clinical development is in resource-poor areas where regulatory hurdles may hamper the timelines Response from authors: This is an excellent point, which we now address in the revised discussion section: “The P2I tool has a number of other limitations, which were described in detail by Young et al. 2 We highlight X specific limitations. First, as a static, deterministic model, it does not take into account possible improvements in product development techniques over time (e.g. R&D efficiencies that reduce costs). Similarly, as a static model, it does not take into account the possibility that candidates may sometimes have to go “backwards” to an earlier phase. For example, once a candidate enters into Phase I there may be bottlenecks that require that the candidate return to a preclinical evaluation stage (e.g., if different formulations need to be re-evaluated or alternate adjuvants need to be tested).” Comment: Step 2 method description and table 3: if in the first run, the authors are using “exactly the same assumptions as P21V2 model”, it is unclear why there is also reference to two additional set of assumptions: the RAP and the BMGF assumptions. Are these readily available? If not, this would make the applicability of P21V2 obsolete especially for other PDPs and PRND vaccine developers with smaller or non-gates supported portfolios. Response from authors: We now discuss the RAP and the Gates Foundation as data sources for P2I.v2; see the revised Methods section. The RAP is not public; the Gates Foundation data are shown in reference 2. Comment: Authors mention that the modified EVI assumptions may be unreliable because they are based only on 2-3 data points and decided to make a pragmatic decision to use only those with 10 data points. This in itself is flawed etc Response from authors: We agree that model should be based on real data, however we do not consider it as meaningful to modify the P2I tool assumptions if such alternative parameters only include 2-3 data points. A better way forward will be to combine these low numbers of alternative data points with additional data points coming from other users of the tool, thereby increasing the overall total number of data points available. Such a combined, increased number of data points can then be used as alternative parameters for the analysis of product portfolios Comment: Probability rates are too generous in the EVI assumptions. Response from authors: Probability rates included are the real success rates obtained at EVI. These high success rates are also not surprising, given that they relate to phase I clinical trials that test for vaccine safety where success rates usually are rather high. Comment: Disease focus of the selected portfolio is quite varied and for some (ie malaria) with multiple targets versus for some only one target. A more detail discussion on the applicability of the tool for the evaluation of 1-2 targets versus >2 targets would be very useful. Response from authors: Wording that had already been included in the article has been slightly modified for clarification. As mentioned in the article, especially for the estimation of expected product launches the P2I tool should be used for an overall analysis of global product candidate portfolio, compared to the analysis of individual organisation´s portfolio only. Such combined global studies will provide far more meaningful data." } ] } ]	1	https://f1000research.com/articles/8-1066
https://f1000research.com/articles/9-79/v1	03 Feb 20	{ "type": "Research Article", "title": "Trends in fertility preference implementation among selected Eastern African countries", "authors": [ "Vincent Otieno", "Alfred Agwanda Otieno", "Anne Khasakhala", "Alfred Agwanda Otieno", "Anne Khasakhala" ], "abstract": "Background: There has been continuous debate among scholars regarding fertility transition in Africa. Two conclusions emerge: slow pace of decline because of weak facilitating social programs and high demand for large families amidst weak family planning programs. Accelerated fertility decline is expected to occur if there is both substantial decline in desired fertility and increased level of preference implementation. Despite these conclusions, there are also emergent exceptions in Africa, even among the Eastern African countries. Our motivation for the study of this region therefore lies in this context. First, the East African countries share some similarities in policy framework. Secondly, Rwanda and Kenya appear as exceptional in the drive towards accelerating further fertility decline. Fertility change therefore in any one country may have implications in the neighbouring country due to the commonalities especially in language, cultural traits, diffusion and spread new models of behaviour. Methods: With the utilization of DHS data, we analyse trends overtime in two specific features that scholars have indicated to slow or increase fertility decline. Using Bongaarts supply-demand framework, we first deduce trends in fertility preferences among women of reproductive age (15-49 years) and second, the extent to which women have been able to implement their fertility preferences during the course of fertility decline and subsequently decomposing these trends. Results: We found that with the rising aggregate of the degree of fertility preference implementation index, continuous declining trends in demand for births and subsequent increases in the contribution made by either or both the wanted fertility and the degree of fertility preference implementation index across categories that fertility transition is certainly on course in all countries albeit at different levels, thanks to the family planning. Conclusions: Family planning programs must therefore be accompanied by rigorous, consistent sensitization and public education.", "keywords": [ "Fertility Preference", "Degree of Preference Implementation Index", "Wanted Fertility", "Eastern Africa" ], "content": "Introduction\n\nOver the last decade, there has been considerable debate among scholars on fertility transition in Africa. Two conclusions emerge: slow pace of decline (Bongaarts, 2017; Bongaarts & Casterline, 2013) because of weak facilitating social programs (Otieno et al., 2016) and high demand for large families amidst weak family planning programs (Bongaarts & Casterline, 2013). Accelerated fertility decline in Sub-Saharan Africa will occur if there is both substantial decline in desired fertility and increased level of preference implementation (Bongaarts, 2017; Bongaarts & Casterline, 2013).\n\nDespite these conclusions, there are also emergent exceptions, such as Rwanda, Ethiopia, Malawi (Bongaarts, 2017; Mahy & Gupta, 2002). In a seminal workshop organized by Committee on Population of the National Academy of Sciences in 2015, there were suggestions that further search for explanations of fertility transition may lie in examinations of specific historical contexts of each population (Casterline & Han, 2017). Our motivation for this study lies in this context. First, the East African countries share some common policy framework. Secondly, Rwanda and Kenya appear exceptional in the drive towards accelerating further fertility decline.\n\nKenyan fertility began to rapidly decline in the 1980s, followed by a stall (1998–2003) and then another phase of decline (2003–2014) recently (KNBS, 2015). Tanzania progressed slowly in decline followed by stall mainly in rural areas (Ezeh et al., 2009; Garenne, 2014) and then further decline within similar timelines as Kenya (Otieno et al., 2016). Uganda has generally been regarded to still be at the pre-transition stage of fertility decline, recently exhibiting some modest declines (Ezeh et al., 2009). Rwanda experienced rapid decline from 2005–2015 (Dhillon & Phillips, 2015; May, 2017) because of the government’s management of the economy and provision of social and health services, including family planning, which are exceptional by regional standards (Dhillon & Phillips, 2015; May, 2017). On the other hand Muhoza et al. (2014) revealed remarkable differences in desired and excess fertility between the four East African countries and between certain communities within these countries.\n\nThe main reason for focus on these countries is that any fertility change in one country may have implications in the neighboring country, because neighbouring regions share common dynamics, including language and cultural traits that permit shared flow of ideas to eventually spread new models of behavior. We analyze trends in two specific features that scholars have indicated will slow or increase in fertility decline. First, trends in fertility preferences among women and secondly, the extent to which women have been able to implement their fertility preferences during the course of fertility decline. Because of heterogeneity in fertility change within countries, we focus on trends by sub-national regions and social class stratification, since understanding the differences in fertility according to socioeconomic status and how these differences evolved over the fertility transition period is of great importance in understanding fertility decline. Conclusions about fertility change have been based on mostly cross-national comparisons based on national data but there has been less attention to sub national differentiation. It has been observed that enormous heterogeneity exists in Kenya, where the wealthy and higher educated people have fertility desires close to replacement level, regardless of religion, while poor, uneducated people, particularly those in Muslim communities, have virtually uncontrolled fertility (Muhoza et al., 2014). On the other hand, in Rwanda the poor, uneducated people have the same desired fertility as their wealthy, educated compatriots, regardless of their religion.\n\n\nConceptual framework\n\nThis study was guided by Bongaarts (1993) modification of the supply-demand framework for fertility analysis developed by Easterlin (1975) and Easterlin & Crimmins (1985). According to Bongaarts, Easterlin’s economic approach is a model of behavioral and biological factors affecting fertility in developing countries. The model consists of three fundamental concepts: demand for children, the potential supply of children, and the momentary and psychic costs of contraception. According to the model, women whose potential supply of births exceeds demand would consider contraception, taking into consideration the costs involved while choosing suitable family planning methods (Montgomery, 1987). In this modification depicted in Figure 1, the fertility outcome measured by the total fertility rate is a function of: supply of births (natural fertility); demand for births (wanted fertility) and degree of preference implementation. The latter in turn is dependent on cost of fertility regulation and cost of unwanted childbearing. The degree of preference implementation is the net result of a decision-making process in which couples weigh the cost of fertility regulation and the cost of unwanted pregnancy.\n\nSource: Bongaarts (1993). The supply-demand framework for the determinants of fertility: An alternative implementation.\n\n\nMethods\n\nAccording to Bongaarts (1993), the relationship between these variables and fertility can be expressed as:\n\n\n\nWhere Fu is unwanted fertility (which can simply be expressed as Fo – Fw), and\n\n\n\nWhere Fn is total natural fertility and Ip is the degree of fertility preference implementation index. Ip ranges from 0 to 1. With full preference implementation, Ip = 1 (which implies that Fu = 0 and F = Fw) and Ip is 0 with no preference implementation (this implies a substantial level of unwanted childbearing and F = Fn). Bongaarts (1993) indicated that Fn is not the same as in total fecundity used in the Bongaarts proximate determinants but is taken to mean fertility level achieved in absence of contraception (see Bongaarts, 1993). Fu is a function of the difference between supply and demand, and the degree of preference implementation. Substitution of Equation 2 from Equation 1 yields\n\n\n\n\n\nWhere C implies is an index ranging from 0 to 1 measuring the reduction in proportional of natural fertility attributable to deliberate birth control.\n\n\n\nWhere U represents the proportion of married women who were practicing contraception at the time of survey. It is measured as the number of married women using contraceptive method to the total number of married women. Substitution of Equation 5 into Equation 4 gives an estimate of Fn, while rearranging Equation 3 gives:\n\n\n\n\nDecomposition of fertility trends\n\nWhen estimates of observed, wanted and natural fertility, as well as the index of implementation are available for two successive points at time t1 and t2 in the same population (Bongaarts, 1993), then a decomposition can be obtained to identify causes of fertility declines in specific populations (Table 1). Again, following Bongaarts’ 1993 formulation, the following variables were used.\n\nThe decline in fertility between t1 and t2 is simply equal to F1 – F2, and this difference can be expressed as a function of the mediating variables by substitution of Equation 3.\n\n\n\nSince the emphasis here is on examining changes in fertility that result from changes in determinants, this equation can be rewritten as\n\n\n\nWhere ΔF, ΔFw, ΔFn and ΔIp represent absolute changes in F, Fw, Fn and Ip, respectively, and Fw, Fn, and Ip with bars represent the average values of Fw, Fn and Ip, respectively. Equation 8 conveniently divides the observed fertility decline ΔF into three components corresponding to each of the three determinants as indicated below (Table 2)\n\nThe above formulation shows that a change in wanted or natural fertility to the observed fertility decline depends on the average level of implementation index. Similarly, the fertility effect from a given change in the index of implementation depends on the average between natural and wanted fertility (Fn – Fw) Table 1 and Table 2). The percentage contribution of each of the determinants to fertility decline can also be obtained by multiplying the ratio of change of each of the determinants to total fertility change by 100.\n\n\nData\n\nThis study utilized cross sectional secondary data gathered overtime by the Demographic and Health Surveys (DHS) collected between 1988 and 2015 for Kenya, Rwanda, Uganda and Tanzania from women of reproductive age (15 to 49 years). DHS data is highly comparable across countries and has been shown to be of high quality. DHS data often computes first the total fertility rates based on data for the three years preceding the survey for age group 15–49 expressed per woman and second the total wanted fertility rates for the three years preceding the survey for age group 15–49 expressed per woman. Total wanted fertility rate is calculated in the same way as the total fertility rate, but only including wanted births. However, an alternative method is also to utilize the formula for the proportion of women age 40–49 in union who want no more births. According to Bongaarts, Fw can be obtained from the equation:\n\n\n\nWhere Fw is the proportion of women who want more children and Wm (40–49) is the proportion of women in union aged from 40 to 49 who want no more births. Bongaarts, however, was cognizant of the fact that indeed there are limitations especially with the information on wanted fertility. The key limitations highlighted here are rationalization, involuntary and voluntary fertility limitation.\n\n\nResults\n\nTable 3, concerning the trends in total fertility rate, wanted fertility rate and fertility preference implementation index for East African countries, shows the results of the application of Bongaarts’ 1993 formula to estimate the degree of fertility preference implementation index for the four East African Countries: Kenya, Rwanda, United Republic of Tanzania and Uganda. Notably, all the countries experienced declines in both fertility and wanted fertility rates (Figure 2). However, the highest change rate in decline occurred in Uganda. Subsequently, over the years as country fertility decline trends continue, Kenya and Rwanda have consistently registered the lowest wanted fertility rates, while Tanzania has since been surpassed by Uganda, as assessed by their respective last surveys. Wanted fertility rates for Tanzania have declined very slowly since 1996. Further, Rwanda recorded the highest rate of change in the desire to have fewer births compared to Kenya while Uganda also recorded the highest wanted fertility parameter values over the same periods until recently when it caught up with Tanzania in the course of decline.\n\nFigure 2 shows the general trends in the degree of fertility implementation index over the years for the respective countries. All countries have improved their implementation indices. Both Kenya and Tanzania experienced a plateau in Ip between 1998 and 2005 perhaps coinciding with the period of fertility decline. Contraceptive use also stagnated or stalled. Rwanda has experienced a rapid rise in Ip since 2005, which is consistent with the beginning of strength of family planning programs in the country. Uganda on the other hand is rising at a slower pace than its counterparts.\n\nThis section presents the effect of both wanted fertility and preference implementation on fertility decline in the four countries based on the decomposition procedure. The analysis is restricted to the periods between 2000 and 2014. Positive values of wanted fertility, natural fertility and implementation index indicate contribution to decline while the negative values indicate tendency to increase fertility. Table 4, concerning the Estimated contribution of preference implementation and wanted fertility to fertility decline shows the change in fertility for the three countries over a period of about a decade. All the countries assessed have witnessed a decline in fertility since the beginning of the century. The highest decline in total fertility rate occurred in Rwanda, while the lowest decline occurred in Tanzania. All the countries subsequently witnessed a decline in wanted fertility rates, but the highest decline occurred in Rwanda. It is also in Rwanda where declines in the demand for children contributed to the highest decline in fertility. Similarly, ability to implement fertility desires has contributed to large declines in fertility across all the countries.\n\nTFR, total fertility rate; Fn, total natural fertility.\n\nExcept for Uganda, in all the other countries, the ability to implement fertility preference contributed to nearly twofold decline in fertility. The results complement the assertion by Bongaarts & Casterline (2013) and Casterline & Han (2017) that acceleration of fertility decline occurs when both demand for children and ability to implement fertility desires occur simultaneously, as in the case of Rwanda and to some extent Kenya. However, the national data mask within country differences. An example is why Tanzania never experienced any large declines in the contribution of wanted fertility to the course of fertility decline as with the case of Kenya and Rwanda.\n\nTable 5, on the estimated Ip and contribution of wanted fertility and preference implementation to fertility change by region, presents variations in fertility preference implementation index and the contribution of wanted fertility and preference implementation to fertility decline at sub-national levels for the four countries. In Kenya, most regions have Ip above 0.85 except for North Eastern region with negative low value. Regions in Rwanda have Ip ranging 0.75 in Western region to 0.96 in Northern region. Tanzania has very wide variation in Ip, with the lowest (0.44) in Pemba south to the highest (1.00) in Kilimanjaro region. However, some regions experience unique results (Dar es Salam, Pwani, Lindi, Mtwara and Zanzibar Town west). Such results might arise due to measurement errors in wanted fertility rate or issues in the measurement of contraceptive use that is utilized in the estimation of natural fertility. However, the low fertility of Mtwara region did not arise from high contraceptive use but from long post-partum amenorrhea. Thus, it may be the case of the effects of other proximate determinants may also influence the estimation of Ip.\n\nBase year: Kenya, 2003; Rwanda, 2005; Tanzania, 2004; Uganda, 2000. Latest survey: Kenya, 2014; Rwanda, 2014-15; Tanzania, 2014-15; Uganda, 2011.\n\nIp < 0; *Ip > 1.\n\nTFR, total fertility rate; Fn, total natural fertility.\n\nThe largest declines in fertility within the decade occurred in Regions of Rwanda (North (2.7-fold), West (2-fold) and East (1.9-fold)). This was followed by Kagera (1.8-fold) and Mbeya (1.7-fold) regions of Tanzania. The next highest decline occurred in South region of Rwanda, Northern of Uganda and Eastern regions Kenya followed by Mtwara region of Tanzania, Nyanza and Rift valley regions of Kenya. Most regions of Tanzania had minimal or no decline at all over the period assessed. The Nairobi region of Kenya, Morogoro and Pemba North regions of Tanzania equally registered no significant change in fertility (Table 3). What is notable is that some regions in Tanzania actually registered increases in fertility. These include the largest urban centre Dar-es-Salaam (−0.8-fold) and Singida (−0.4-fold) in Tanzania mainland and Zanzibar North (−0.7-fold), and Zanzibar South (−1.3-fold) in Zanzibar Island. The low decline in fertility rate as well as observed increase in fertility in some regions of Tanzania may explain why there has been slow change in average fertility indicators for Tanzania at the national level. Fertility preference is by large a key contributor to the reduction of fertility, notably in Tanzania. The regions with largest contribution by change in wanted fertility to fertility decline were Manyara at 708% and Mtwara at 226%. This was followed by the East region of Rwanda at 204%, Ruvuma of Tanzania at 177% and the West and North regions of Rwanda with 155% and 137%, respectively. Others with significant contributions were Iringa, Kilimanjaro and Lindi regions of Tanzania. Thus, the major contribution to fertility decline in Tanzania across many sub-regions is the change in fertility preferences.\n\nIn Kenya, Rwanda and Uganda, the ability to implement fertility desires Ip was the greatest contribution to fertility decline for most of the regions. The contribution of Ip was highest in South and Kigali regions in Rwanda, Pwani and Lindi regions of Tanzania, where Ip contributed to over a twofold decline in fertility. Notwithstanding this, the Ip contributed least to fertility decline mostly in regions of Tanzania. These regions include Manyara, Rukwa and Iringa. Bongaarts’ formulation equation rule is that the index of implementation of fertility can only fall between zero and one. However, this has not been the case in some segments of the populations within this study. It calls for a reflection on some regions that registered erratic indices. These regions actually posted indices values either below zero or above unity. The regions were Pemba South of Tanzania North Eastern region of Kenya at −0.09-fold, while the indices beyond unity values were the Pwani, Mtwara, Lindi and Dar es Salaam in Tanzania, which therefore calls for revisiting the formula to include the effect of other related proximate determinants of fertility decline, acting as a normalization of sort.\n\nTable 6 highlights the contributions made by the wanted fertility and the degree of fertility preference implementation index to the change in fertility based on two key socioeconomic variables namely, the type of place of residence and the level of educational attainment of the women. In all regions, Ip increased with increase in level of education. Ip was also higher in urban areas compared to rural areas in all regions. In Kenya and Rwanda, fertility decline was higher in rural areas and among women with lower or no education. Tanzania posted mixed and unique results. In general, the degree of fertility preference implementation index was higher among women of urban resident as well as among women with higher education.\n\nBase year: Kenya, 2003; Rwanda, 2005; Tanzania, 2004; Uganda, 2000. Latest survey: Kenya, 2014; Rwanda, 2014-15; Tanzania, 2014-15; Uganda, 2011.\n\nTFR, total fertility rate; Fn, total natural fertility; Fw, wanted fertility.\n\nIn Rwanda, both changes in wanted fertility and ability to implement fertility desires contributed to fertility decline across all the sub groups. However, in Kenya it was the ability to implement fertility desires that made major contribution to fertility decline across most of the subgroups. Uganda had almost similar results to Rwanda except that the contribution was lower. Similarly, Tanzania had similar results to Kenya, though with lower contribution values.\n\n\nDiscussion\n\nIn this study, we focused on the sub-national regions (provinces) of East African countries, analyzing trends in fertility preferences among women and the extent to which these women have been able to implement these fertility preferences during the course of declining fertility in these countries. The main reason was that fertility change in one country may have implications in the neighboring country because neighboring regions share common language and cultural traits that permit shared flow of ideas and eventual spread of new models of behavior. In Kenya and Rwanda, the most important contributor to fertility decline by region, place of residence and socio-economic groups is the ability to implement fertility desires (Ip). The same is true for Uganda; however, Tanzania posted mixed results by different groups.\n\nRwanda has had the largest fertility decline due to the contributions by both changes in the wanted fertility and ability to implement fertility desires. The results highlight one core result: fertility declines faster when both wanted fertility and ability to implement desired fertility occur simultaneously, as in the case of Rwanda. Secondly, we find enormous heterogeneity in fertility change within countries. The largest heterogeneity existed in Tanzania and lowest in Rwanda. As indicated by Muhoza et al. (2014), there are not only remarkable differences in desired fertility between the four East African countries and between certain communities within these countries, but also the degree to which the different communities are able to implement their desires. These differences may reflect cultural differences in reproductive behavior, as well as effect of development and population density which create resource needs pressures. For example, for Kenya, Tanzania and Uganda, regions with high agricultural potential, high population density and the highest densities of development inputs, have high implementation indexes and the lowest desired family sizes.\n\nThe results here concur with Bongaarts & Casterline (2013), who found that although each country has a unique fertility trajectory, fertility transitions share similarities. Countries typically have high and relatively stable fertility during the pre-transitional period which comprises most of human history (Bongaarts & Casterline, 2013). Once the transition starts, the pace of fertility decline in the first one or two decades following the onset is usually faster than in later decades. In any given country, today’s level of fertility may be a function of the pre-transitional level of fertility, the timing of the onset of the fertility transition, and the pace of fertility change. As noted earlier, conventional transition theory predicts that fertility levels are inversely related to socioeconomic development indicators (Bongaarts & Casterline, 2013).\n\nThere is some caution that may be made in the estimation of Ip as evident in the North Eastern region of Kenya and some regions of Tanzania. The Ip is dependent on development of a region, but more importantly closely associated with extent of unwanted fertility and hence unmet need for contraception. A close assessment into the cultural factors reveal that Muslim dominated areas may appear to have low degree of Ip. However, this may be confounded by other factors. Zanzibar Town West region has a high Ip while Pemba south has low Ip, whereas the North Eastern region of Kenya has low levels of contraception utilization. These regions—dominated by cultural dogmas and doctrines, especially religious beliefs—exhibit rationalized feedbacks and indeed prone to even non-numeric responses (Casterline & Han, 2017). Furthermore, the level of Ip may be dependent on program reach, confirming that fertility change must be based on the concurrent change in both women’s preferences and ability to implement those preferences.\n\n\nConclusions and recommendations\n\nIt is now clear that with the rising aggregate level of the degree of fertility preference implementation index, continuous declining trends in demand for births and the subsequent increases in the contribution made either singly or jointly by the wanted fertility and the degree of fertility preference implementation index across the various categories indicates that fertility transition is certainly on course in all countries albeit at different levels. In sum, the evidence examined here shows that the pattern of fertility decline in the region is indeed unique. In addition, the relatively slow pace and level of development in the region implies that the cost of raising children has remained low compared to other developing regions and the benefits of having offspring remain substantial in the subsistence economies, which characterizes the East African countries. This is also true of a majority of the Sub-Saharan African countries in general (Bongaarts, 2017).\n\nThe dominant mediums of diffusion of knowledge and information among populations, such as the public media and urbanization, though still very low, are expected to increase their influence. The future pace of fertility decline in the region will therefore likely be slower than the pace of decline in other regions at comparable times from the transition onset, unless special interventions are undertaken (Bongaarts & Casterline, 2013). This is a result of the duration it has taken the region to get to where it is compared to the countries that went through transition in the earlier years.\n\nThere is, however, an additional policy option to accelerate fertility decline. Investing in family planning programs to provide information about and access to contraception in order to permit control of reproductive lives and avoiding unplanned pregnancies. These countries in general are characterized among the ones with low contraception (Alkema et al., 2013). The key cause of an unmet need for contraception is that contraception is either unavailable or often too costly to the consumers and sometimes the population is ignorant of the existence or usage of contraception. In addition, there are significant non-economic costs such as health concerns, social disapproval, and spousal resistance, as well as unnecessary medical barriers requiring higher-level expertise for utilization (Casterline & Sinding, 2000). The unmet need is responsible for most of the unsatisfied demand subsequently aggravating unplanned pregnancies.\n\nThese family planning programs therefore must be accompanied by rigorous, consistent sensitization and public education campaigns through media among other modes of communication, which in turn trigger demand for contraception in anticipation of lower desired family size by diffusing new ideas about the benefits of smaller families and the role of women (Bongaarts, 2017). The degree of implementation is expected to take in values between zero and one. However, there is evidence of values greater than one as well as negative values. This points to a limitation in the frontiers of knowledge with regards to this study. Due to these glaring violations further research is recommended to assess how well the fertility preference implementation index is suited to measuring fertility success.\n\n\nData availability\n\nThe datasets analyzed during the current study are available in the MEASURE DHS repository (http://www.measuredhs.com). Access to the dataset requires registration and is granted to those that wish to use the data for legitimate research purposes. A guide for how to apply for dataset access is available at: https://dhsprogram.com/data/Access-Instructions.cfm. The DHS datasets used in the present study are listed in Table 3.", "appendix": "References\n\nAlkema L, Kantorova V, Menozzi C, et al.: National, regional, and global rates and trends in contraceptive prevalence and unmet need for family planning between 1990 and 2015: a systematic and comprehensive analysis. Lancet. 2013; 381(9878): 1642–1652. PubMed Abstract \| Publisher Full Text\n\nBongaarts J: The supply-demand framework for the determinants of fertility: An alternative implementation. Popul Stud. 1993; 47(3): 437–456. Publisher Full Text\n\nBongaarts J: Africa's unique fertility transition. Popul Dev Rev. 2017; 43(S1): 39–58. Publisher Full Text\n\nBongaarts J, Casterline J: Fertility transition: is sub-Saharan Africa different? Popul Dev Rev. 2013; 38(Suppl 1): 153–168. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCasterline JB, Han S: Unrealized fertility: Fertility desires at the end of the reproductive career. Demogr Res. 2017; 36: 427–454. Publisher Full Text\n\nCasterline JB, Sinding SW: Unmet need for family planning in developing countries and implications for population policy. Popul Dev Rev. 2000; 26(4): 691–723. Publisher Full Text\n\nDhillon RS, Phillips J: State capability and Rwanda's health gains. Lancet Glob Health. 2015; 3(6): e308–e310. PubMed Abstract \| Publisher Full Text\n\nEasterlin RA: An economic framework for fertility analysis. Stud Fam Plann. 1975; 6(3): 54–63. PubMed Abstract\n\nEasterlin RA, Crimmins EM: The fertility revolution: A supply-demand analysis. University of Chicago Press. 1985. Reference Source\n\nEzeh AC, Mberu BU, Emina JO: Stall in fertility decline in Eastern African countries: regional analysis of patterns, determinants and implications. Philos Trans R Soc Lond B Biol Sci. 2009; 364(1532): 2991–3007. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarenne M: Trends in marriage and contraception in sub-Saharan Africa: A longitudinal perspective on factors of fertility decline. DHS Analytical Studies No. 42. Rockville, Maryland, USA : ICF International. 2014. Reference Source\n\nKNBS: Health Survey (KDHS). 2014. Kenya National Bureau of Statistics. 2015.\n\nMahy M, Gupta N: Trends and differentials in adolescent reproductive behavior in sub-Saharan Africa. DHS Analytical Studies No. 3. Calverton, Maryland, USA: ORC Macro. 2002. Reference Source\n\nMay JF: The Politics of Family Planning Policies and Programs in sub-Saharan Africa. Popul Dev Rev. 2017; 43(S1): 308–329. Publisher Full Text\n\nMontgomery MR: A new look at the easterlin “synthesis” framework. Demography. 1987; 24(4): 481–496. PubMed Abstract\n\nMuhoza DN, Broekhuis A, Hooimeijer P: Variations in desired family size and excess fertility in East Africa. International Journal of Population Research. 2014; 2014. Publisher Full Text\n\nOtieno AA, Amani HK, Makbel A: POPULATION DYNAMICS AND SOCIAL POLICY.2016. Reference Source" }	[ { "id": "63692", "date": "10 Jun 2020", "name": "John Bongaarts", "expertise": [ "Reviewer Expertise Demography", "Fertility", "Family planning" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper sheds light on the causes of fertility transition in selected Eastern African countries. The authors rely on a decomposition method I proposed in 1993 which estimates the roles of three factors in fertility decline: 1) decline in wanted fertility, 2) increase in preference implementation and 3) change in natural fertility. Results show that the rise in the implementation index is the main driver of fertility decline. Although the study does not directly assess the impact of family planning programs, the authors note that these programs aim to help women in implementing their reproductive preferences and disseminate information about the benefits of contraception and smaller families.\nThe text is clearly written and the analysis is technically competent. Two minor edits:\nThe total fertility rate is represented by three different abbreviations: TFR, F, and Fo. This is confusing and should be fixed.\n\nThe first paragraph of the results section makes reference to Figure 2. This should be Table 2.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "128259", "date": "19 Apr 2022", "name": "Collins C Iwuji", "expertise": [ "Reviewer Expertise Clinical epidemiology", "population health", "HIV", "STIs", "planetary health" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study investigated the interaction between wanted fertility and fertility preference implementation in shaping fertility trends in 4 countries in Eastern Africa. This is a detailed analysis that explored this phenomenon both at the national and subnational level highlighting heterogeneity within and between countries.\nAbstract\nThe sentence summarising the results in the abstract is very long and difficult to follow. I suggest the authors revise this and use shorter sentences for clarity\nMain article General comments\nThe authors should be consistent when describing the different types of fertility – wanted fertility, natural fertility, and total fertility – and should use consistent notations throughout the article. This makes for ease of reading and understanding.\nResults\nThe first paragraph in the results section where Figure 2 was mentioned should have been Table 3.\n\nThe authors did well to explore other socio-economic factors that could explain the observed fertility trend in the four countries studied, such as place of residence and educational attainment.\n\nIn the analysis or perhaps in the discussion, other country-level factors that could shape decisions around fertility should have been explored, for example, whether education is free in the countries and the availability of free health care. I suggest the authors explore these in the discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-79
https://f1000research.com/articles/9-71/v1	31 Jan 20	{ "type": "Study Protocol", "title": "What are the outcomes of core decompression in patients with avascular necrosis? Protocol for a systematic review", "authors": [ "Octavian Andronic", "Haitham Shoman", "Ori Weiss", "Vikas Khanduja", "Haitham Shoman", "Ori Weiss", "Vikas Khanduja" ], "abstract": "Background: Core decompression is a hip preserving surgical procedure that is used to treat avascular necrosis (AVN) of the femoral head. The eventual clinical and radiological outcome following this procedure is varied in literature. Also, the time to a total hip replacement (THR) from the index procedure and the percentage of patients subsequently undergoing a THR is controversial. Furthermore, there are multiple surgical methods along with multiple augmentation techniques and various classification and staging systems described. The purpose of this systematic review, therefore, is to analyse the outcomes following decompression only, excluding any augmentation techniques for non-traumatic AVN of the femoral head. Methods: This protocol is being developed in line with the PRISMA-P guidelines. The search strategy includes articles from Medline, Embase, Google Scholar, CINHAL and Cochrane library. The review and screening will be done by two independent reviewers. Review articles, editorials and correspondences will be excluded. Articles including patients with sickle cell disease and with core decompression where augmentation is used will be excluded. The risk of bias and quality of articles will be assessed using the Joanna Briggs Institute Critical Appraisal Checklist for the different study designs included. Discussion: This study will be a comprehensive review on all published articles having patients with AVN of the femoral head and undergoing core decompression surgery only. The systematic review will then define the outcomes of the core decompression surgery based on clinical and radiological outcomes. Each outcome will include the different stages within it and finally, the total mean time to THR will be calculated. This will then be followed by assessing the cumulative confidence in evidence from all the data collected using the GRADE tool.\n\nRegistration: This systematic review is registered in the International Prospective Register for Systematic Reviews and Meta-analysis (PROSPERO) under the registration number: CRD42018100596", "keywords": [ "Core decompression", "hip", "avascular necrosis", "hip preservation", "femoral head", "avn." ], "content": "Introduction\n\nOsteonecrosis or avascular necrosis (AVN) of the femoral head is a challenging condition that eventually leads, in the majority of cases, to a total hip replacement (THR)1–3. The patients affected by the condition are usually young and therefore may require revision surgery and multiple further procedures4. The aetiology is varied and includes multiple conditions that can lead to a reduced blood supply in the femoral head: oral corticosteroids, systemic lupus erythematosus, binge consumption of alcohol, Gaucher disease, sickle cell anaemia, trauma, thrombosis amongst others5. Furthermore, staging systems for AVN are different across the literature and pose a significant problem in assessing surgical indications and stratifying outcome6. The most common classification systems used are: Ficat7/ Modified Ficat8; “University of Pennsylvania”/Steinberg6; and ARCO (Association Research Circulation Osseous)9.\n\nCore decompression is a common surgical procedure that has been used earlier on in the disease process to decrease the intraosseous pressure in the femoral head, relieve pain and potentially re-establish blood flow. Furthermore, multiple augmentation techniques have recently been described that seem to significantly improve the outcome following this procedure10,11.\n\nHowever, the eventual outcome and time to THR following this procedure remains controversial12–16. It is also not clear whether a mechanical decompression alone is sufficient and efficient in all stages of AVN in order to prevent progression and delay the need of a THR. To the best of our knowledge, the largest published systematic review on this subject included only four studies for analysis17.\n\nThe purpose of this study, therefore, was to assess the outcomes of core decompression without any augmentation for nontraumatic AVN of the femoral head.\n\n\nMethods/design\n\nThe Preferred Reporting Items for Systematic Reviews and Meta-analysis – Protocol (PRISMA-P) guidelines will be used to develop the protocol of the study. The manuscript will then be developed using the PRISMA statement and flowchart. This systematic review is registered in the International Prospective Register for Systematic Reviews and Meta-analysis (PROSPERO) under the registration number: CRD42018100596.\n\nThe search for articles will include several databases, Medline, Embase, Google Scholar and the Cochrane library. There will be no restriction on dates and articles will be included from inception. The search strings will include articles looking at patients with AVN and having core decompression and they will then be combined using the Boolean terms AND/OR. A total of eight combinations of the following keywords will be used: “femoral head” with “osteonecrosis”, “avascular necrosis”, “aseptic necrosis”, “avn” with the terms - “core decompression” or “surgery”.\n\nFirst, a blinded and independent process of selection based on title and abstract will be made by two authors (OA and HS). Secondly, a thorough analysis of eligible studies was performed by evaluating full texts. Studies will then be screened according to the inclusion and exclusion criteria (Table 1). Articles included would be in English language, looking at those suffering from AVN and those who had core decompression only. Reviews, editorials and commentaries will be excluded. The PICO tool is used to formulate the research question. The participants will be everyone with no restriction to age, race or gender, the intervention is core decompression, there will be no comparator, and the outcomes will include clinical and radiological (Table 2).\n\nThe selected articles will then be exported to Mendeley reference manager software and all duplicates will be removed electronically and manually. The final number of included articles will then be assessed for full text review and data will be extracted based on a pre-determined set of variables. Two reviewers (AO and OW) will assess and screen and if there is any discrepancy, a third more senior reviewer (VK) will be invited to advice until consensus is met between all authors. Data extracted will then be pulled into a spreadsheet with the pre-determined variables on Microsoft Excel v16.21.\n\nThe extracted data will based on the following table headings: author, study setting (country and year), number of included hips, average follow up, gender percentages, average age, mean Body Mass Index (BMI), preliminary diagnosis (primary etiology), stage of disease, surgical technique, clinical outcome (with preoperative and postoperative results where applicable), radiological outcome and time to THR.\n\nThe risk of bias and quality of studies will be evaluated using the Joanna Briggs Institute Critical Appraisal Checklist18 for each study design due to its rigor in assessing the methodological integrity of studies. The outcomes of core decompression will include clinical and radiological outcomes. In addition, the total mean time for hip replacement among all the studies will be estimated. Based on the quality of studies included, a meta-analysis might be conducted across the studies if there was limited heterogeneity in the data.\n\nIn cases of changes in the existing protocol that significantly would affect the accuracy of data, scope of the investigation, or scientific quality of the study, edits will be performed and a newer version that would be in accordance with the final systematic review, will be provided and published.\n\nThe systematic review is planned to be submitted upon completion to an orthopaedic peer-reviewed journal with global audience and then uploaded under copyright conditions to further dissemination platforms, such as Research Gate and others.\n\n\nStudy status\n\nOngoing.\n\n\nDiscussion\n\nClassification systems, outcome measures and reporting systems are highly variable amongst studies assessing and reporting the outcome of core decompression for AVN of the femoral head. From distinct classifications (Ficat or its modification, Steinberg, ARCO) to varied clinical scores (Harris Hip Score/D’Aubigne/Visual Analogue Scale), all have been described and used in the literature.\n\nFurthermore, the concept of “procedural success” is not absolute. Whilst most studies consider the absence of radiological progression of disease to be the main finding that suggests success, other authors interpret clinical improvement as success, even in the presence of radiological deterioration. Therefore, the heterogeneity of interpretation of success in the studies extracted may not allow for a uniform representation. As such, the results will be represented in separate categories, based on the classification system that was used and divided into radiological progression and another category of clinical improvement.\n\nThe strengths of our study will be represented by the largest patient pool and rigorous exclusion criteria that will be used. Any collateral influence of aetiology (traumatic), systemic disease (sickle cell crisis) or technique heterogeneity (presence of augmentation or bone grafts) will be excluded. Also, there will be a tenacious stratification based on stage of the disease even in the presence of a variety of classification systems. Ultimately, the following questions will be evaluated and answered:\n\n1) Does core decompression accomplish postoperative pain relief/clinical or functional improvement?\n\n2) In what percentage of the patients does core decompression achieve a cessation of radiological progression?\n\n3) What percentage of patients undergoing core decompression without augmentation, ultimately require a THR?\n\n4) What is the average time to requiring a THR following core decompression?\n\n\nData availability\n\nNo data is associated with this work.\n\nFigshare: PRISMA-P checklist for ‘What are the outcomes of core decompression in patients with avascular necrosis? Protocol for a systematic review’, https://doi.org/10.6084/m9.figshare.11720289.v319.\n\n\nAuthor information\n\nVK (MD, MA, MSc, FRCS(Orth)) is a Consultant Orthopaedic Surgeon at Addenbrooke’s - Cambridge University Hospital, England, UK. VK is also an Associate Lecturer at the University of Cambridge and the Associate Editor at the Bone and Joint Journal. He is also the Chair of SICOT Education Academy and Chair of the UK’s non-arthroplasty hip registry. OA (MD) is VK’s research fellow and orthopaedic surgery resident at the Balgrist University Hospital in Zurich, Switzerland. OW (MD) is VK’s research fellow and orthopaedic surgery resident at the Meir Medical Center, Kfar-Saba, Israel. HS (MD, DIC, MPH) is also VK’s research fellow and a Global Surgery Research Fellow at Harvard Medical School, Boston, MA, USA.", "appendix": "References\n\nYoon BH, Mont MA, Koo KH, et al.: The 2019 Revised Version of Association Research Circulation Osseous Staging System of Osteonecrosis of the Femoral Head. J Arthroplasty. 2019; pii: S0883-5403(19)31101-5. PubMed Abstract \| Publisher Full Text\n\nYoon BH, Jones LC, Chen CH, et al.: Etiologic Classification Criteria of ARCO on Femoral Head Osteonecrosis Part 2: Alcohol-Associated Osteonecrosis. J Arthroplasty. 2019; 34(1): 169–174.e1. PubMed Abstract \| Publisher Full Text\n\nYoon BH, Jones LC, Chen CH, et al.: Etiologic Classification Criteria of ARCO on Femoral Head Osteonecrosis Part 1: Glucocorticoid-Associated Osteonecrosis. J Arthroplasty. 2019; 34(1): 163–168.e1. PubMed Abstract \| Publisher Full Text\n\nBergh C, Fenstad AM, Furnes O, et al.: Increased risk of revision in patients with non-traumatic femoral head necrosis. Acta Orthop. 2014; 85(1): 11–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShah KN, Racine J, Jones LC, et al.: Pathophysiology and risk factors for osteonecrosis. Curr Rev Musculoskelet Med. 2015; 8(3): 201–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSultan AA, Mohamed N, Samuel LT, et al.: Classification systems of hip osteonecrosis: an updated review. Int Orthop. 2019; 43(5): 1089–1095. PubMed Abstract \| Publisher Full Text\n\nJawad MU, Haleem AA, Scully SP: In brief: Ficat classification: avascular necrosis of the femoral head. Clin Orthop Relat Res. 2012; 470(9): 2636–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith SW, Meyer RA, Connor PM, et al.: Interobserver reliability and intraobserver reproducibility of the modified Ficat classification system of osteonecrosis of the femoral head. J Bone Joint Surg Am. 1996; 78(11): 1702–6. PubMed Abstract \| Publisher Full Text\n\nSchmitt-Sody M, Kirchhoff C, Mayer W, et al.: Avascular necrosis of the femoral head: inter- and intraobserver variations of Ficat and ARCO classifications. Int Orthop. 2008; 32(3): 283–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArbeloa-Gutierrez L, Dean CS, Chahla J, et al.: Core Decompression Augmented With Autologous Bone Marrow Aspiration Concentrate for Early Avascular Necrosis of the Femoral Head. Arthrosc Tech. 2016; 5(3): e615–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMa Y, Wang T, Liao J, et al.: Efficacy of autologous bone marrow buffy coat grafting combined with core decompression in patients with avascular necrosis of femoral head: a prospective, double-blinded, randomized, controlled study. Stem Cell Res Ther. 2014; 5(5): 115. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith SW, Fehring TK, Griffin WL, et al.: Core decompression of the osteonecrotic femoral head. J Bone Joint Surg Am. 1995; 77(5): 674–80. PubMed Abstract \| Publisher Full Text\n\nBozic KJ, Zurakowski D, Thornhill TS: Survivorship analysis of hips treated with core decompression for nontraumatic osteonecrosis of the femoral head. J Bone Joint Surg Am. 1999; 81(2): 200–9. PubMed Abstract \| Publisher Full Text\n\nMarkel DC, Miskovsky C, Sculco TP, et al.: Core decompression for osteonecrosis of the femoral head. Clin Orthop Relat Res. 1996(323): 226–33. PubMed Abstract \| Publisher Full Text\n\nSimank HG, Brocai DR, Brill C, et al.: Comparison of results of core decompression and intertrochanteric osteotomy for nontraumatic osteonecrosis of the femoral head using Cox regression and survivorship analysis. J Arthroplasty. 2001; 16(6): 790–4. PubMed Abstract \| Publisher Full Text\n\nYoon BH, Lee YK, Kim KC, et al.: No differences in the efficacy among various core decompression modalities and non-operative treatment: a network meta-analysis. Int Orthop. 2018; 42(12): 2737–2743. PubMed Abstract \| Publisher Full Text\n\nRajagopal M, Balch Samora J, Ellis TJ: Efficacy of core decompression as treatment for osteonecrosis of the hip: a systematic review. Hip Int. 2012; 22(5): 489–93. PubMed Abstract \| Publisher Full Text\n\nAromataris E, Fernandez R, Godfrey CM, et al.: Summarizing systematic reviews: methodological development, conduct and reporting of an umbrella review approach. Int J Evid Based Healthc. 2015; 13(3): 132–40. PubMed Abstract \| Publisher Full Text\n\nAndronic O, Shoman H, Weiss O, et al.: PRISMA-P checklist and flow chart for ‘What are the Outcomes of Core Decompression in Patients with Avascular Necrosis? Protocol for a Systematic Review’. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11720289.v3" }	[ { "id": "60053", "date": "23 Mar 2020", "name": "Tony Andrade", "expertise": [ "Reviewer Expertise Hip preservation surgery", "including arthroscopic and open hip procedures." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have submitted a study protocol for a study in which they set out to determine the outcomes of core decompression (without any augmentation procedure) for non-traumatic avascular necrosis of the femoral head. They aim to carry out a systematic review in line with PRISMA-P guidelines, with the review and screening carried out by two independent reviewers.\nTheir study design and the described methods are completely appropriate.The outcomes will be based on clinical and radiological outcomes, and the mean time to Total Hip Replacement will also be calculated. Finally the GRADE tool will be used to assess the cumulative confidence in evidence from all the data collected.\nPrevious authors have concluded from their meta-analyses, which mostly employed augmentation treatments, that as they could not find any differences in the rates of THA conversion and radiologic progression across all core decompression modalities and non-operative treatment, their results question the assumption that core decompression changes the natural course of avascular necrosis of the femoral head. The authors need to consider this.\nOne of the potential limitations with previous meta-analyses has been the relatively low numbers of cases, and so the authors of this study need to be able to aim for higher numbers in their review.\nPrevious studies have concluded with a call for very large scale randomised controlled trials to confirm the effectiveness of core decompression in itself. This further systematic review may well add to this call.\nI have also added in two citations that the authors should review, as these further inform the subject.\nThe study is a very valuable one that aims to inform on the outcomes of decompression alone (without augmentation techniques) for AVN of the femoral head. This is a very important topic in the hip preservation setting and these results will add significant value to the existing literature. I therefore fully support this study, with only the minor points I have detailed above.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "61725", "date": "26 Mar 2020", "name": "Andreas Roth", "expertise": [ "Reviewer Expertise Orthopaedic surgery", "osteoporosis", "femoral head necrosis." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe aim of the preliminary study is to examine the results of core decompression in avascular necrosis as part of a systematic review. The authors have presented a complete study protocol for this. In principle, study design and methods are suitable to achieve the goal.\nBecause of the heterogeneity of the present work, they want to evaluate the results in different categories, which are based on the respective classification system and differ in terms of radiological progression and the improvement of the clinical findings.\nThe authors must be aware that the number of patients described is relatively small. I took the liberty to list the literature I know.\nIn fact, the papers available differ with regard to the primary endpoint. But the classification systems are also different. Last but not least, the surgical techniques used are different, and sometimes not exactly specified. These are the potential limitations of this work.\nUltimately, however, it is of great interest for all those who deal with this clinical picture to obtain such results. Therefore, I would like to support this work and advocate the indexing of the project.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-71
https://f1000research.com/articles/8-1845/v1	04 Nov 19	{ "type": "Research Article", "title": "Relationship between postpartum depression and lactation status at a Japanese perinatal center: A cross-sectional study", "authors": [ "Shunji Suzuki" ], "abstract": "Background: Some studies have demonstrated that breastfeeding can protect mothers from postpartum depression; therefore, we examined the association between postpartum depression and lactation status at one month after delivery at a Japanese perinatal center. Methods: We reviewed the obstetric records of all (total 809) nulliparous healthy women with vaginal singleton delivery at 37-41 weeks’ gestation at our institute between July 2018 and June 2019. A face-to-face interview with the women was conducted on admission for delivery to ask whether or not they hoped to perform exclusive breastfeeding for their babies, and an additional interview was conducted one month after delivery to ask about their feeding methods currently. Maternal mental status was examined based on the scores using the Edinburgh Postnatal Depression Scale (EPDS), and women with EPDS scores of ≥9 points were regarded as ‘positive screening’. Results: 592 women (73.1%) hoped to perform exclusive breastfeeding for their babies on admission. Of these, at one month, 442 (74.7%) performed exclusive breastfeeding, while 150 (25.3%) performed mixed or artificial feeding. The average EPDS scores and the incidence of EPDS scores ≥9 points in the women performing exclusive breastfeeding were 4.3 ± 3.6 and 14.3% (63/442), respectively. They did not differ from those in the women performing mixed or artificial breast feeding [4.2 ± 3.7, p = 0.60 and 13.3% (20/150), p = 0.78]. Conclusion: Development of postpartum depression does not seem to be associated with incomplete breastfeeding at our hospital, and therefore there are other risk factors indicated in the development of postpartum depression.", "keywords": [ "postpartum depression", "lactation status", "exclusive breastfeeding", "the Edinburgh Postnatal Depression Scale", "Japan" ], "content": "Introduction\n\nExclusive breastfeeding for the first 6 months of life has been recommended because of important health, medical, social, and developmental benefits to both mothers and babies1. Postpartum depression has been recognized as the leading medical complication among new mothers2,3. To date, some risk factors for postpartum depression, such as personal and family factors, socioeconomic status, support from other family members and personal plans for furthering careers, have been examined4–8. Some studies have demonstrated that breastfeeding can protect mothers from postpartum depression and are starting to clarify which biological and psychological processes may explain this protection9–11. In addition, a short duration of breastfeeding has been reported to be associated with the development of postpartum depression11.\n\nIn Japan, the breastfeeding rate at Japan’s baby-friendly hospitals (BFHs) at one month of age has been reported to be more than 75%12; however, inconsistent knowledge of breastfeeding benefits and inappropriate hospital practices has been reported to be associated with the increased use of infant formula and reduced breastfeeding duration, although the national breastfeeding rates had been higher than other countries of similar health status13. Unfortunately, only 50% of women who delivered at Japanese Red Cross Katsushika Maternity Hospital, a non-BFH institute, have performed exclusive breastfeeding for their babies at one month after delivery14,15. Recently, in Japan, the population of elderly and/or high-risk pregnant women has been increased, and the rate of exclusive breastfeeding may be expected to decrease4. To examine the necessity of breastfeeding promotion in relation to maternal mental status, we examined the association between lactation status and postpartum depression at one month after delivery in Japanese women.\n\n\nMethods\n\nThe protocol for this analysis was approved by the Ethics Committee of the Japanese Red Cross Katsushika Maternity Hospital (approval number, K2018001). Written informed consent was obtained from each woman to participate in this study at the first meeting, i.e. before birth.\n\nWe reviewed the obstetric records of all nulliparous healthy women (n = 809) with vaginal singleton delivery at 37–41 weeks’ gestation at Japanese Red Cross Katsushika Maternity Hospital between July 2018 and June 2019.\n\nWe excluded cases of multiparous women, multiple births, cesarean deliveries, mothers with a habit of smoking and/or drinking, mothers with pregnancy depression, mothers without partners mothers whose babies are low birth weight, and mothers whose babies were admitted to the neonatal intensive care unit (NICU) because they have been already reported to be associated with the prevalence of exclusive breastfeeding and/or postpartum depression4,11,16–18.\n\nA face-to-face interview was conducted with the women on admission for delivery to ask them whether or not they hoped to perform exclusive breastfeeding for their babies at the delivery room of the hospital, and an additional interview was conducted one month after delivery to ask about their feeding methods at that time at the outpatient examination room of the hospital during routine check-up appointments.\n\nMaternal mental status was examined, at one month after delivery, based on the scores of the questionnaires of the Edinburgh Postnatal Depression Scale (EPDS) at the same time of the interview. In this study, women with the EPDS scores of ≥9 points were regarded as ‘positive screening = 50% possibility of depression’, according to the results of previous observations in Japan by Okano et al.19,20.\n\nData are presented as the mean ± SD or number (%). SPSS Statistics software version 20 (IBM Csorp., Armonk, NY, USA) was used for statistical analyses. For statistical analysis, the Χ2 test for categorical variables and Student’s t-test for continuous variables were used. Differences with p < 0.05 were considered significant.\n\n\nResults\n\nOn admission, 592 women (73.1%) hoped to perform exclusive breastfeeding for their babies and who met the conditions to be considered in the current study. Of these, 442 (74.7%) performed exclusive breastfeeding at one month, while 150 (25.3%) performed mixed or artificial feeding (mixed feeding: 296, artificial feeding: 24). There were no significant differences in maternal age between the two groups (exclusive breastfeeding: 32.4 ± 6.1 years; mixed or artificially feeding: 32.8 ± 6.4 years; p = 0.11).\n\nThe average EPDS scores and the incidence of EPDS scores of ≥9 points in the women performing exclusive breastfeeding were 4.3 ± 3.6 and 14.3% (63/442), respectively. These did not differ from those in the women performing mixed or artificial feeding [4.2 ± 3.7, p = 0.60 and 13.3% (20/150), p = 0.78]. In addition, the average EPDS score and the incidence of EPDS scores of ≥9 points in the women performing exclusive artificial feeding was 4.0 ± 3.2 and 8.3% (2/24), respectively.\n\n\nDiscussion\n\nAlthough the rate of exclusive breastfeeding may be low in our institute, it did not seem to be related to the development of postpartum depression. The current results seemed to be contrary to those in some previous studies indicating that breastfeeding can protect mothers from postpartum depression9–11.\n\nTo date, lower plasma oxytocin levels leading to incomplete breastfeeding have been reported to be associated with the development of postpartum depression. In a recent study by Lara-Cinisomo et al.10, for example, lower levels of plasma oxytocin were observed in women who had stopped breastfeeding and had postpartum depression by two months postpartum. The influence of synthetic oxytocin on a new mother’s well-being has been also reported previously10,21,22. Oxytocin is released across the breastfeeding cycle, and oxytocin release has observed to exhibit a temporary anxiolytic-like calming effect on postpartum maternal mood disturbances21. Therefore, oxytocin is believed to mediate a calming effect on postpartum mood in primiparous mothers with breastfeeding.\n\nHowever, in this study, exclusive breastfeeding did not contribute to the prevention of postpartum depression significantly. The mental status of mothers considered to have low levels of oxytocin associate with incomplete breastfeeding seemed to be stable. Therefore, in social environments and/or clinical characteristics of pregnant Japanese women, there may be some serious risk factors for postpartum depression other than the status of breastfeeding, such as personal and family factors, socioeconomic status, support from other family members and personal plans for furthering careers4–8. For example, in our recent Japanese study that asked mothers’ biggest worry at two weeks after delivery, only 10% reported anxiety about breastfeeding8. Although we believe that Japan is not a poor country, recently there are some morbid pregnant women who have been reduced to poverty23.\n\nWe understand the small sample of the current study is a serious limitations. A study in women who gave birth at BFHs may have totally different results than the current results. In addition, although the EPDS has been the most widely used screening tool for postpartum depression in maternity and child services in various countries throughout the world, a EPDS high score does not mean the presence of postpartum depression19,20,24,25.\n\nIn conclusion, development of postpartum depression does not seem to be associated with incomplete breastfeeding in Japanese women at our institute. Consequently, there must be other risk factors associated with the development of postpartum depression. A further larger study is needed to clarify these factors.\n\n\nData availability\n\nFigshare: Breastfeeding and EPDS, https://doi.org/10.6084/m9.figshare.9925070.v126\n\nThis project contains the following underlying data:\n\n- Dataset 1. Raw data for maternal age, breastfeeding methods and EPDS score recorded from 592 women who hoped to perform exclusive breastfeeding for their babies.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe author wishes to thank all patients for their collaboration.\n\n\nReferences\n\nKramer MS, Kakuma R: Optimal duration of exclusive breastfeeding. Cochrane Database Syst Rev. 2002; 1(8): CD003517. PubMed Abstract \| Publisher Full Text\n\nYeaton-Massey A, Herrero T: Recognizing maternal mental health disorders: beyond postpartum depression. Curr Opin Obstet Gynecol. 2019; 31(2): 116–119. PubMed Abstract \| Publisher Full Text\n\nPearlstein T, Howard M, Salisbury A, et al.: Postpartum depression. Am J Obstet Gynecol. 2009; 200(4): 357–364. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTakeda S, Takeda J, Murakami K, et al.: Annual Report of the Perinatology Committee, Japan Society of Obstetrics and Gynecology, 2015: Proposal of urgent measures to reduce maternal deaths. J Obstet Gynaecol Res. 2017; 43(1): 5–7. PubMed Abstract \| Publisher Full Text\n\nHonjo K, Kimura T, Baba S, et al.: Association between family members and risk of postpartum depression in Japan: Does \"who they live with\" matter? -The Japan environment and Children's study. Soc Sci Med. 2018; 217: 65–72. PubMed Abstract \| Publisher Full Text\n\nMuchanga SMJ, Yasumitsu-Lovell K, Eitoku M, et al.: Preconception gynecological risk factors of postpartum depression among Japanese women: The Japan Environment and Children's Study (JECS). J Affect Disord. 2017; 217: 34–41. PubMed Abstract \| Publisher Full Text\n\nIwata H, Mori E, Tsuchiya M, et al.: Predictors of depressive symptoms in older Japanese primiparas at 1 month post-partum: A risk-stratified analysis. Jpn J Nurs Sci. 2016; 13(1): 147–155. PubMed Abstract \| Publisher Full Text\n\nSuzuki S: Mother's biggest worry at 2 weeks after delivery (in Japanese). Clin Gynecol Obstet (Tokyo). 2017; 71(11): 1107–1111.\n\nFigueiredo B, Dias CC, Brandão S, et al.: Breastfeeding and postpartum depression: state of the art review. J Pediatr (Rio J). 2013; 89(4): 332–338. PubMed Abstract \| Publisher Full Text\n\nLara-Cinisomo S, McKenney K, Di Florio A, et al.: Associations Between Postpartum Depression, Breastfeeding, and Oxytocin Levels in Latina Mothers. Breastfeed Med. 2017; 12(7): 436–442. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDias CC, Figueiredo B: Breastfeeding and depression: a systematic review of the literature. J Affect Disord. 2015; 171: 142–154. PubMed Abstract \| Publisher Full Text\n\nYoda T, Takahashi K, Yamauchi Y: Japanese trends in breastfeeding rate in baby-friendly hospitals between 2007 and 2010: a retrospective hospital-based surveillance study. BMC Pregnancy Childbirth. 2013; 13: 207. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInoue M, Binns CW, Otsuka K, et al.: Infant feeding practices and breastfeeding duration in Japan: A review. Int Breastfeed J. 2012; 7(1): 15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSuzuki S, Hirohata S, Uriu K, et al.: Cesarean delivery as a factor promoting exclusive breastfeeding in Japan. J Matern Fetal Neonatal Med. 2013; 26(17): 1762–1763. PubMed Abstract \| Publisher Full Text\n\nSuzuki S: Maternal age and breastfeeding at 1 month after delivery at a Japanese hospital. Breastfeed Med. 2014; 9(2): 101–102. PubMed Abstract \| Publisher Full Text\n\nKaneko A, Kaneita Y, Yokoyama E, et al.: Factors associated with exclusive breast-feeding in Japan: for activities to support child-rearing with breast-feeding. J Epidemiol. 2006; 16(2): 57–63. PubMed Abstract \| Publisher Full Text\n\nRowe-Murray HJ, Fisher JR: Operative intervention in delivery is associated with compromised early mother-infant interaction. BJOG. 2001; 108(10): 1068–1075. PubMed Abstract \| Publisher Full Text\n\nPilch D: [The influence of birth modus on the emotional state of the mother, bonding, and the newborn's neurobehavioural state]. Pomeranian J Life Sci. 2015; 61(3): 249–256. PubMed Abstract\n\nOkano T, Nomura J, Kumar R, et al.: An epidemiological and clinical investigation of postpartum psychiatric illness in Japanese mothers. J Affect Disord. 1998; 48(2–3): 233–240. PubMed Abstract \| Publisher Full Text\n\nOkano T: [Perinatal depression--recent topics]. Nihon Rinsho. 2007; 65(9): 1689–1693. PubMed Abstract\n\nNiwayama R, Nishitani S, Takamura T, et al.: Oxytocin Mediates a Calming Effect on Postpartum Mood in Primiparous Mothers. Breastfeed Med. 2017; 12: 103–109. PubMed Abstract \| Publisher Full Text\n\nGu V, Feeley N, Gold I, et al.: Intrapartum Synthetic Oxytocin and Its Effects on Maternal Well-Being at 2 Months Postpartum. Birth. 2016; 43(1): 28–35. PubMed Abstract \| Publisher Full Text\n\nSuzuki S: Economic problems and mental disorders in Japanese pregnant women. J Clin Med Res. 2015; 7(5): 367. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCox JL, Murray D, Chapman G: A controlled study of the onset, duration and prevalence of postnatal depression. Br J Psychiatry. 1993; 163: 27–31. PubMed Abstract \| Publisher Full Text\n\nCox JL, Holden J, Henshaw C: Perinatal Mental Health; The Edinburgh Postnatal Depression Scale (EPDS) Manual 2nd Edition. Glasgow, UK. RCPsych Publications (Royal College of Psychiatrists), 2014. Reference Source\n\nSuzuki S: breastfeeding and EPDS. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9925070.v1" }	[ { "id": "58361", "date": "24 Jan 2020", "name": "Hiroko Iwata", "expertise": [ "Reviewer Expertise postpartum depression", "maternity nursing", "midwifery", "childrearing support for older primiparous mothers" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMethods:\nHow did the author control confounding factors? Postpartum depression is reported to have associations with multiple factors, as the author stated in “Introduction”. The author performed univariate analysis (i.e., χ2 test and Student’s t-test), not multivariate analysis. If so, results should be interpreted carefully, or some statement as a study limitation is recommended.\nResults:\nIt is noteworthy and interesting that we could observe a trend of higher score of EPDS and more women with EPDS score of > 9 among exclusive breastfeeding group of women, though the results were non-significant. How did the author interpret those results?\nDiscussion:\nThe first sentence “Although the rate of exclusive breastfeeding may be low in our institute…” does not make sense for readers. The author should contextualize its meaning within the Japanese culture.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5176", "date": "30 Jan 2020", "name": "Shunji Suzuki", "role": "Author Response", "response": "Response to the reviewer,Many thanks for your careful reading of the manuscript. We appreciate your comments very much. Thank you very much for your suggestions. Based on the queries, we have re-written the many parts of the manuscript.We have added ‘to control confounding factors’ in the Methods. In addition, we have added a limitation concerning the confounding factors.We have arranged the first paragraph of the Discussion as suggested. Thank you very much for considering our paper, again.Sincerely yours,Shunji Suzuki, MDDepartment of Obstetrics and Gynecology,Japanese Red Cross Katsushika Maternity Hospital5-11-12-2 Tateishi, Katsushika-ku, Tokyo 124-0012 JapanTel: +81-3-3693-5211Fax: +81-3-3694-8725e-mail: czg83542@mopera.ne.jp" } ] } ]	1	https://f1000research.com/articles/8-1845
https://f1000research.com/articles/9-59/v1	29 Jan 20	{ "type": "Case Report", "title": "Case Report: Primary ganglioneuroblastoma occurrence in lower thoracic spine", "authors": [ "Masuma Islam", "Muhammad Khurram Zia", "Syeda Ifra asad", "Syed Zawahir Hassan", "Osama Salam", "Saeed Mazhar", "Gohar Jawad", "Taha Nafees", "Masuma Islam", "Muhammad Khurram Zia", "Syeda Ifra asad", "Osama Salam", "Saeed Mazhar", "Gohar Jawad", "Taha Nafees" ], "abstract": "Ganglioneuroblastoma is a neural tissue neoplasm, which is derived from the neural crest cells. It is mostly seen in the pediatric population but is very rarely found in the lower thoracic spine. Here, we report a rare case of ganglioneuroblstoma occurrence in the lower thoracic spine. A 2.5-year-old boy presented with spinal compression symptoms and on magnetic resonance imaging, a mass was identified over T10 to L1. The tumor showed round blue cells and mature ganglion cells with hypermitotic activity. Immunohistochemical synaptophysin and neurofilament staining was positive, confirming the diagnosis. The patient showed significant improvement after surgical excision of the tumor. This is the first reported case of ganglioneuroblastoma in the lower thoracic spine that was successfully treated in Pakistan.", "keywords": [ "Ganglioneuroblastoma", "Embryonal tumor", "Juvenile brain tumor" ], "content": "Introduction\n\nGanglioneuroblastoma is a tumor of peripheral neuroblastic tissue that arises from the neural ectodermal cells. Ganglioneuroblastoma is a variant of neuroblastic tumor, which encompasses neuroblastoma, ganglioneuroblastoma and ganglioneuroma. The most commonly seen is the neuroblastoma, which contributes 97% of the neuroblastic tumors. Among pediatric cancers, the neuroblastoma is the third most common cancer after brain cancer and leukemias1. Among neuroblastic tumors, neuroblastoma is the least differentiated, ganglioneuroma is the most differentiated and ganglioneuroblastoma is the intermediate between them. Neuroblastoma histology includes primitive neuroblasts, ganglioneuroblastoma includes both primitive neuroblasts and ganglion cells and ganglioneuromas include mature Schwann cells and ganglion cells as the most differentiated variant. Neuroblastic tumors are seen in the adrenal gland, para spinal neural tissue, chest, neck, spine, intra-abdominal areas and the presentation of these tumors is highly dependent on their location and metastasis. Ganglioneuroblastomas are rarely seen in the spinal column2.\n\nThe authors present here the first case of primary intraspinal ganglioneuroblastoma of the thoracic and lumbar spine in Pakistan. A histopathological study helped the authors to establish the diagnosis. Surgical excision is the treatment of choice for both cerebral and spinal ganglion cell tumors. Radiotherapy is given for residual disease, recurrence, or progression. We report a 2.5-year-old boy who achieved improvement after surgical excision.\n\n\nCase presentation\n\nA 2.5-year-old South Asian boy resident of Karachi presented to the emergency room with an inability to urinate for the last seven days. This was followed by sudden paralysis of both lower limbs of two days’ duration., resulting in the patient being completely unable to sit, stand or walk. The patient had a history of difficulty in walking and standing for the prior six months.\n\nA neurological examination revealed sensory loss below the T12 vertebra and a bilateral increase in muscle tone. Using the Medical Research Council scale for muscle power, power was 0/5 on the left side and 1/5 on the right side upon flexion and extension. Babinski reflexes were normal on both sides, indicating an upper motor lesion. The child had no past history of birth asphyxia, hypertension, diabetes mellitus or tuberculosis and their vaccination history was normal, with vaccines administered at routine time points. The child had no past history of trauma or other constitutional symptoms. The child was afebrile. An MRI scan was carried out, which showed a large homogenous enhancing lesion spreading from the T8 to L1 vertebrae. The intra-spinal region was completely adherent to the dural coverings of the dorsal cord and dural sleeves of nerve roots. Moreover, the abdominal region of cord covered the retroperitoneal structures, including the renal fascia, kidneys, bowels and para-spinal area, extending up to the aorta and adjacent vascular structures.\n\nThe surgical plan included a five level laminectomy, which was then performed immediately, and the whole segment was reconstructed after complete resection of the spinal lesion. The lesion was badly adherent to the cord roots. Two cord roots were compromised and the cerebrospinal fluid leak was fixed. Operative findings were of a soft elastic, moderately vascularized, exophytic tumor, extending into the extradural region of the spine. The tumor had extradural growth and encircled the tissue around the spinal cord, extending into the retroperitoneal area as well. The biopsy sample was sent for histopathology reporting, which revealed a tumor consisting primarily of nodules and sheets of mature looking ganglion cells with abundant Schwannian stroma with diffusely present round blue cells, hyperchromatic nuclei and scanty cytoplasm. These cells showed increased mitotic activity as well. Hematoxylin and eosin staining showed the tumor to be neurofilament and synaptophysin positive. According to the International Neuroblastoma Staging System, the patient had a stage 4 tumor as metastasis to other organs was found. In conclusion, the histopathology report confirmed the diagnosis of an intermixed ganglioneuroblastoma. and the patient was assigned to intensive modulation radiation therapy with a dose of 18 grays for six months.\n\nAt the follow-up appointment two weeks later, a physical examination was carried out and power was found to be improved on the right side, measuring 2/5, and on the left side, measuring 1/5. The patient returned two months later for a follow-up appointment, when a detailed neuro-physical examination was carried out and power was increased, measuring 3/5 on the left side and 2/5 on the right side. A timeline of events from initial symptoms to post-surgery follow up meeting is shown in Figure 1.\n\nER, emergency room; MRI, magnetic resonance imaging.\n\n\nDiscussion\n\nGanglioneuroblastomas are a rare type of neuroblastic tumor that are composed of a varying proportion of undifferentiated neuroblast and mature ganglionic cells3. Ganglioneuroblastoma is a pediatric tumor and is uncommon in the adult population. The histology of ganglioneuroblastomas involves tumor cells derived from the mantle layer of the embryological spinal cord that populate the embryological sympathetic ganglia and adrenal medulla. Ganglioneuroblastoma histology includes undifferentiated small round cells in all stages of neuronal differentiation2,4. These embryological neural crest cells may mature to become Schwann cells or remain undifferentiated, called neuroblasts5,6.\n\nGanglioneuroblastomas are subdivided into two categories (nodular and intermixed) and both have significance in terms of prognosis, along with age of disease presentation, disease pathogenesis, genetics, nuclear maturation and grading. Ganglioneuroblastomas have both ganglionic and neuroblast cells, which both have the potential to become malignant7. In the nodular type of ganglioneuroblastoma, there is ganglioneuromatous stroma around nodules of neuroblasts and in the intermixed type, there are small numbers of neuroblasts with ganglioneuromatous stroma.\n\nSymptoms of ganglioneuroblastomas are highly dependent on the primary location of the tumor and metastasis. Symptoms include abdominal distension, weight loss, irritability, focal neurological deficits and opsoclonus-myoclonus. The most common presentation of ganglioneuroblastoma is abdominal distension. Survival rates in infancy are almost 91% and decline as the age of presentation increases. Ganglioneuroblastomas show no gender predominance and are seen equally among males and females. Ganglioneuroblastomas predominantly present in the first decade of life7 and are very uncommon among adults, with most affected patients being under five years of age8. Genetics plays an important role in the pathogenesis of neuroblastic tumors, although sporadic cases are also common.\n\nGanglioneuroblastomas predominantly involve the adrenal gland, although other sites such as retroperitoneal ganglion, posterior mediastinum and pelvis are also involved. Ganglioneuroblastomas are likely to be seen among the sympathetic ganglia and therefore often occur in the paramedian region in the neck, pelvis and head9. Ganglioneuroblastoma arising in the lower thoracic spine is one of the rare presentations of this tumor2,10,11. The least common sites for ganglioneuroblastomas to occur are the thymus, cauda equina, lung, spinal column and kidney. The spinal column is a rare site for ganglioneuroblastomas to occur and it is quite possible that the tumor extended intraspinally from the mediastinum. In the spinal column, ganglioneuroblastomas are seen as extradural lesions with a dumbbell shape.\n\nGanglioneuroblastomas are associated with various diseases including Von Recklinghausen disease, Hirsch sprung disease, DiGeorge syndrome and Beckwith-Weidman syndrome7,12. The most common site of ganglioneuroblastoma metastasis is the bone, which is called Hutchinson’s disease and presents as limping and unexplained irritability. Other metastatic locations include the liver and skin8. Definitive treatment involves resection of the tumor, and radiotherapy is also included in cases of high-grade tumors.\n\n\nConclusion\n\nGanglioneuroblastoma is a neuroblastic tumor that is mostly seen in the pediatric population. The lower thoracic spine is a rare location for a tumor and it is very important to include ganglioneuroblastoma in the differential diagnosis when investigating lower thoracic spine masses. With proper investigative approaches, ganglioneuroblastoma can be diagnosed early and can be managed accordingly, decreasing morbidity and mortality in patients.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "References\n\nShohet JM, Nuchtern JG: Epidemiology, pathogenesis, and pathology of neuroblastoma. Hentet fra. 2016. Reference Source\n\nSingh SK, Srivastava C, Ojha BK, et al.: Ganglioneuroblastoma of the conus: a rare and aggressive tumor. Neurol India. 2010; 58(6): 966–8. PubMed Abstract \| Publisher Full Text\n\nTripathy K, Misra A, Gouda AK, et al.: Primary intraspinal ganglioneuroblastoma of the thoracic spine: a rare case report. Indian J Pathol Microbiol. 2012; 55(4): 535–7. PubMed Abstract \| Publisher Full Text\n\nKleihues P: Pathology and genetics of tumors of the nervous system. In: Kleihues P. Editor. World Health Organization Classification of Tumors. Lyon: IARC Press. 2000; 141–4.\n\nJoshi VV: Peripheral neuroblastic tumors: pathologic classification based on recommendations of international neuroblastoma pathology committee (Modification of shimada classification). Pediatr Dev Patholy. 2000; 3(2): 184–99. PubMed Abstract\n\nMatthay KK: Neuroblastoma: a clinical challenge and biologic puzzle. CA Cancer J Clin. 1995; 45(3): 179–92. PubMed Abstract \| Publisher Full Text\n\nLonergan GJ, Schwab CM, Suarez ES, et al.: Neuroblastoma, ganglioneuroblastoma, and ganglioneuroma: radiologic-pathologic correlation. Radiographics. 2002; 22(4): 911–34. PubMed Abstract \| Publisher Full Text\n\nFatimi SH, Bawany SA, Ashfaq A: Ganglioneuroblastoma of the posterior mediastinum: a case report. J Med Case Rep. 2011; 5(1): 322. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamaswamy B, Bhandarkar AM, Menon SS, et al.: Ganglioneuroblastoma of Skull Base. J Clin Diagn Res. 2015; 9(8): MD01–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKikuchi K, Saito M: Ganglion-cell tumor of the filum terminale: immunohistochemical characterization. Tohoku J Exp Med. 1999; 188(3): 245–56. PubMed Abstract \| Publisher Full Text\n\nPatnaik A, Mishra SS, Mishra S, et al.: Primary extradural spinal ganglioneuroblastoma: a case report. Turk Neurosurg. 2014; 24(2): 253–5. PubMed Abstract \| Publisher Full Text\n\nAlessi S, Grignani M, Carone L: Ganglioneuroblastoma: Case report and review of the literature. J Ultrasound. 2011; 14(2): 84–8. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "61414", "date": "25 Mar 2020", "name": "Bidyut Prava Das", "expertise": [ "Reviewer Expertise Immunogenetics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is an excellent article with full progressive history of the patient and every possible investigation has been considered to arrive at the diagnosis. The authors have also discussed different criteria to exclude the other possible differential diagnosis to arrive at the final diagnosis. It will help the practitioners for future reference.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "5369", "date": "31 Mar 2020", "name": "Zawahir Hassan", "role": "Author Response", "response": "Thank you so much for your time." } ] }, { "id": "61942", "date": "17 Apr 2020", "name": "Yuvaraja Murugan", "expertise": [ "Reviewer Expertise Spinal surgery", "Orthopaedics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA very challenging case and its management has been described in a clear manner by the authors. However, the absence of any supporting evidence - i.e., radiological or clinical pictures, histopathology pictures - severely limits the utility of this case report to another clinical practitioner.\nI recommend that the authors provide the entire gamut of clinical and radiological images along with histopathology to enrich the value of this article.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-59
https://f1000research.com/articles/9-58/v1	28 Jan 20	{ "type": "Research Article", "title": "Current routines for antibiotic prophylaxis prior to transrectal prostate biopsy: a national survey to all urology clinics in Sweden", "authors": [ "Johan Styrke", "Sven Resare", "Karl-Johan Lundström", "Patrick Masaba", "Christofer Lagerros", "Pär Stattin", "Sven Resare", "Karl-Johan Lundström", "Patrick Masaba", "Christofer Lagerros", "Pär Stattin" ], "abstract": "Background: The risk of infection after transrectal ultrasound (TRUS)-guided prostate biopsies is increasing. The aim of the study was to assess the use of antibiotic prophylaxis for prostate biopsy in Sweden. Methods: All public and private urology clinics reporting to the National Prostate Cancer Register of Sweden received a survey on TRUS-biopsy prophylaxis. Results: Of the 84 clinics surveyed, 76 replied (90%). If no risk factors for infection were present, a single dose of ciprofloxacin 750 mg was used by 50 clinics (66%). Multiple doses of ciprofloxacin 500 or 750 mg (n=14; 18%) or a single dose of trimethoprim-sulfamethoxazole 160/800 mg (n=7; 9%) were other common prophylaxes. Most clinics gave the prophylaxes immediately before the biopsy (n=41; 54%). Urine dipstick was used by 30 clinics (39%) and rectal enema by six (8%). In patients with high risk of infection, the survey mirrors a large variety of regiments used. Conclusions: The preference to use a single dose of ciprofloxacin 750 mg is in accordance with the Swedish national guidelines for patients with a low risk of infection. Better compliance to the guideline recommendation to use a urine dipstick would probably increase the number of patients classified as having an increased risk of infection. Being classified as a high-risk patient should lead to an extended duration of antibiotic prophylaxis, however, the variety of regimens used in the high-risk group reflects an inability to treat these patients in a standardized fashion and also highlights a need for more clear-cut guidelines. Pre-biopsy identification of high-risk patients is an important issue to tackle for the urologic clinics in order to reduce the number of infections.", "keywords": [ "Prostate biopsy", "Antibiotic prophylaxis", "Prostate cancer diagnostics", "Survey" ], "content": "Introduction\n\nCommon side effects following transrectal ultrasound-guided prostate biopsy (TRUS-biopsy) include urinary tract infections (UTI) sometimes leading to hospitalization1. In Sweden, about 6% of the patients have a prescription of antibiotics during the first month following TRUS-biopsy and about 1% are hospitalized2. The infection rate can be reduced by the use of prophylactic antibiotics, or bowel cleansing with povidone-iodine3–7. The use of antibiotic prophylaxis is underscored by the European, American and new national Swedish guidelines8–10. The most commonly used antibiotics are fluoroquinolones in various regimens3,11,12.\n\nA rising problem is the presence of drug-resistant bacteria leading to increased post-biopsy infection rates13, especially fluoroquinolone-resistant Escherichia coli is problematic and have been assessed to explain over 40% of TRUS-biopsy infections in the USA14. In Europe, fluoroquinolone resistance is found in 8–46% of E. coli isolates, the former figure is the Swedish rate15,16. Resistance to trimethoprim-sulfamethoxazole is found in about 20% of isolates in Sweden16. The rising resistance calls for studies aiming at identifying new suitable antibiotics or new ways of reducing the use of fluoroquinolones. There is also a need for strict adherence to guidelines to avoid overuse of antibiotics and to better identify risk groups for infection, i.e. patients with indwelling catheter, patients with a urine dip-stick positive for nitrite, patients with previous urinary tract infection, diabetes or immunosuppressive treatment according to the national Swedish guidelines, first published in April 201410. Patients with risk factors for infection show slightly higher infection rates than those without2. A recent study concluded that adherence to the European Association of Urology (EAU) guidelines on prophylactic antibiotics safely can reduce the use of antibiotics and lower resistance rates17.\n\nThe primary aim of the present study was to describe the type and timing of antibiotics used prior to TRUS-biopsy in Sweden in low- and high-risk patients and to investigate to what extent urine dipstick, urine culture and rectal enema is used. The secondary aims were to investigate if the antibiotic strategy has changed during 2006–2014 and to compare adherence to the Swedish national guidelines between university hospital departments, non-university hospital departments and private practitioners.\n\n\nMethods\n\nAn electronic survey (available as Extended data18) was distributed to all of the hospitals and outpatient urology clinics reporting to the national Swedish National Prostate Cancer Register (NPCR). The register captures 98% of all prostate cancer cases when compared to the mandatory national cancer register19. The web-based Information Network for CAncer registers in Sweden (INCA) platform was used for reporting. Recipients of the survey were the trained staff reporting to the NPCR or the heads of department if contact with the staff could not be established. In one case, where neither of these recipients could be reached, the survey was distributed to a urologist known by the authors at the clinic in question. The questionnaire comprised six questions concerning current standard prophylaxis in patients with and without risk factors for infection, time of administration, the use of urine dipstick, urine culture or rectal enema prior to TRUS-biopsy during 2006–2014 (Table 1–Table 3). All of the questions were followed by a question regarding if, and when, strategies had been altered during 2006–2014 (Table 4). The questionnaires were distributed 2014-11-26 and after up to three reminders per e-mail, the last response was collected 2015-11-03.\n\nThe table displays the answers from urology departments in Sweden in late 2014 / early 2015 regarding their current use of antibiotic prophylaxes prior to prostate biopsy.\n\nThe table displays the answers from urology departments in Sweden in late 2014 / early 2015 regarding their current routines in identifying high-risk patients with urinary tract infections prior to prostate biopsy and also if rectal enema is used.\n\nThe table displays the answers from urology departments in Sweden in late 2014/early 2015 regarding their current use of antibiotic prophylaxes prior to prostate biopsy in high-risk patients.\n\nThe table displays the answers from urology departments in Sweden in late 2014 / early 2015 concerning their changes in routines prior to prostate biopsy during 2006-2014 with respect to drug of choice, time of administration, use of urine dipstick, use of urine culture, use of rectal enema and duration of treatment.\n\naTen had reduced the amount of prophylaxis from multiple- to single dose regimens, one had changed the dose of a single ciprofloxacin administration, six had changed from trimethoprim-sulfamethoxazole to ciprofloxacin, three had changed from ciprofloxacin to trimethoprim-sulfamethoxazole, one had changed from amoxicillin to ciprofloxacin and one had changed regimen but could not recall how.\n\nbThirteen had changed from early administration to administration immediately prior to the biopsy and two had done the opposite change, one had changed but could not recall how.\n\ncTen had introduced urine dipstick as routine on all patients, one had stopped using urine dipstick and instead used urine culture on all patients, one had changed but could not recall how.\n\ndThree had introduced routine urine culture during the study period.\n\neOne had quit using enema, two had decided to introduced enema in 2015.\n\nfThree had adopted to the guidelines in 2014, one had introduced a checklist in 2012, two had adopted a “more active strategy” and one had changed strategy but could not recall how.\n\nData from the survey was downloaded into Microsoft Excel 2011 (Microsoft Corp., Redmond, WA) and exported to SPSS Statistics 23 (SPSS Inc., Chicago, IL) for further analysis. Standard descriptive statistics were used to present the results. A comparison was conducted between university hospital departments, non-university hospital departments and private practitioners for adherence to the national Swedish guidelines10 defined as using a single dose of prophylaxis to the low-risk group, using multiple dose regimens to the high-risk group and analysing urine dipstick for nitrite prior to biopsy. The Likelihood Ratio-test (G2) was used. Statistical significance was defined as p<0.05. All missing data are presented in Table 1–Table 4.\n\nPatient data were not investigated in the present study. An ethics approval was still obtained from the local ethics committee in Umeå, no 2016-228/31. The committee approved the research project according to the application.\n\n\nResults\n\nThe survey was sent to 84 recipients and answers were obtained from 76 of the clinics (90%) (Figure 1). Of these, seven were university hospital departments, 47 were non-university public hospital departments, two were hospital departments owned by a private company, one was a hospital department owned by a foundation and 19 were private practitioners. De-identified survey responses are available as Underlying data18.\n\nNPCR, The Swedish National Prostate Cancer Register.\n\nIn patients without risk factors for infection the most frequently used antibiotic was a single dose of ciprofloxacin 750 mg used by 50 clinics (66%). The second and third most common choices were multiple doses of ciprofloxacin 500 mg or 750 mg used by 14 clinics (18%) and a single dose of trimethoprim-sulfamethoxazole 160/800 mg used by seven clinics (9%). The prophylaxes were distributed immediately before or immediately after the biopsy in 41 (54%) and 12 (16%) of the clinics respectively, more than one hour before the biopsy in 8 (11%) and both before and after the biopsy in 12 clinics (16%) (Table 1). A single dose of ciprofloxacin 750 mg distributed as recommended by the national guidelines—immediately before or more than one hour before the biopsy—was used by 39 clinics (46%).\n\nUrine dipstick was used by 30 clinics (39%), urine culture as routine for low-risk patients by three clinics (4%) and rectal enema by six clinics (8%) (Table 2). In the question regarding the high-risk group, the results were mixed; 19 clinics used prolonged prophylaxis (>1 dose, <4 days), 13 clinics (17%) used treatment ≥4 days without a urine culture and 11 clinics (15%) utilized the same strategy as for the low-risk patients (of which seven only used single dose prophylaxis) (Table 3).\n\nThere were 10 clinics (13%) that reduced the amount of antibiotic prophylaxis from multiple- to single-dose regimens during the study period; six (8%) had changed from trimethoprim-sulfamethoxazole to ciprofloxacin and three (4%) had done the opposite. A further 13 (17%) had changed from early administration to administration immediately prior to the biopsy and 10 (13%) had introduced urine dipstick as routine on all patients (Table 4).\n\nWhen comparing university hospital departments, non-university hospital departments and private practitioners for adherence to guidelines, no significant differences were found (p=0.8) (Figure 2).\n\nThe figure displays summarized answers from urology departments in Sweden in late 2014/early 2015 regarding their current adherence to guidelines for antibiotic prophylaxes prior to prostate biopsy. Adherence to guidelines is defined as using a single dose of prophylaxis to the low-risk group, using multiple dose regimens to the high-risk group and analysing urine dipstick for nitrite prior to biopsy.\n\n\nDiscussion\n\nIn the present study, the self-reported antibiotic prophylaxis standard patterns for transrectal prostate biopsies in Sweden 2006–2014 are reported. The web-based survey covered 90% of clinics diagnosing prostate cancer in Sweden.\n\nThe first published study to investigate the clinical routines for TRUS-biopsies in Sweden in 2011 addressed the regular procedure in a standard case scenario and did not account for patients with elevated infection risk20. The preferred antibiotic prophylaxis consisted of ciprofloxacin at the time of biopsy (64%) in patients without risk factors for infection, which is comparable to our results (70%). However, dosage and exact timing of administration (i.e. immediately before or after biopsy) were not inquired. Trimethoprim-sulfamethoxazole was the second most common alternative in the respective studies (12% vs 9%). The similarities between the studies are not surprising given the overlapping time periods. The use of fluoroquinolones as standard is supported by two meta-analyses showing that fluoroquinolones have significantly better effects compared with placebo in all included trials3,7. Other antibiotic agents that have been investigated alone or in addition to fluoroquinolones include trimethoprim-sulfamethoxazole, gentamicin, fosfomycin, piperacillin-tazobactam, amikacin, ceftriaxone, amoxicillin-clavulanate and meropenem3,6,7,20–24. Most regiments appear to reduce the risk of infection but there is a lack of sufficiently powered randomized trials comparing fluoroquinolones to other antibiotic classes7. The most recent meta-analysis, however, concludes that the use of augmented antibiotics might be beneficial25. None of these alternatives have been evaluated in a Swedish setting with relatively low resistance. Diversification of substances for antibiotic prophylaxis depend on information about efficacy obtained from randomized trials complemented with local bacterial resistance patterns and possibly information obtained by the use of rectal swabs—a strategy supported by a number of studies26,27.\n\nAccording to the present study, most urology departments give the antibiotic prophylaxis immediately before the prostate biopsy. This strategy is supported by a study by Lindstedt et al. prospectively comparing 1322 biopsies in 1161 patients from two nearby hospitals. At one of the hospitals, 750 mg ciprofloxacin was given two hours prior to the biopsy and at the other hospital the same prophylaxis was given immediately before the biopsy. The results revealed no significant differences between the groups in both of which hospital admission for febrile urinary tract infection (UTI) occurred in less than one per cent of the cases28. Owing to the low number of febrile UTI (n=12 in total) the possibility that the study was underpowered to find a small significant difference cannot be ruled out; the study was also not randomized, making the level of evidence lower. These results have been extrapolated to determine the timing also of trimetoprim-sulfamethoxazole prior to TRUS-biopsy in Sweden. Future randomized trials are needed to investigate this issue further. Two meta analyses have not shown significant differences in risk of infection when comparing single versus multiple doses of antibiotics3,7.\n\nA urine dipstick test is useful for bacteriuria screening if the results of both nitrites and leukocyte-esterase are negative29; however, a small number of false-negative tests will occur28. Prior urine bacterial culture does not seem to be advantageous in patients without risk factors for infection28,30. However, a more stringent implementation of a dipstick urine sample may allow identifying more patients at risk for infection. As a consequence, patients would have to undergo urine culturing prior to TRUS-biopsy which may also possibly reduce the risk for prophylactic antibiotic failure.\n\nIn patients with risk factors for infectious complications the clinical practice varied in our study with the most widespread strategy being an extended duration of antibiotic prophylaxis (42%). This is somewhat in line with the EAU guidelines on urological infection favouring an individual approach in patients at risk8. The variety of prophylactic regimens also reflects the difficulties in identifying these patients in a standardised fashion. Risk factors for infection outlined in the national Swedish guidelines include a positive urine dip stick or urine culture, previous febrile infections following prostate biopsy, previous urinary tract infections or bacterial prostatitis, diabetes, immunosuppression or an indwelling bladder catheter10. Other risk factors that have been proposed are presence of fluoroquinolone resistant E. coli, old age, previous prostate biopsy, hospitalization prior to biopsy and non-adherence to antibiotic prophylaxis6,13. The rising bacterial resistance to fluoroquinolones in the rectal flora over time has led to a rise in infectious complications1,6,27. Patients who have previously been treated with fluoroquinolones have been identified as a group at risk for harbouring bacteria with fluoroquinolone resistance31. The current national Swedish guideline does not address this risk group explicitly. A systematic and thoroughly taken patient history accounting for the presence of these risk factors must be emphasized as a measure of importance to find patients at high risk of infection. A better adherence to the guidelines for low-risk patients may similarly reclassify patients to the high-risk group. There is a need for studies aiming at reducing the risk of infection as well as reducing the amount of antibiotics used in the high-risk group.\n\nThat only a minority of the clinics adhere to the national Swedish guidelines might be explained by the fact that the guidelines were first introduced in April 2014. Hopefully the figures will improve over the years to come.\n\nThe main weakness of the study is that the answers to the questionnaire reflect the official policy of each clinic and are not provided by individual doctors, as strategies may differ between doctors within the same clinic. It is, however, likely that there are local routines used by most urologists—at least regarding the low-risk group as there are well established guidelines for this group10. The antibiotic strategy in high-risk patients probably varies more and the lack of clear-cut national guidelines may lead to a more individual approach by urologists. Regarding the questions about regimens changes during 2006–2014, there is a risk of recall bias. The survey was constructed by the authors before being sent out and is not validated. It was conducted in late 2014; it is, however, likely that most clinics still use the strategies shown in the survey because no major changes in the national or European guidelines for prostate cancer have emerged since then. The strength of the study is that it includes 90% of all clinics conducting prostate biopsies in Sweden. The results only apply to Swedish conditions but due to the high response rate the internal validity is assessed to be high.\n\nIn conclusion, the preference to use a single dose of ciprofloxacin 750 mg is in accordance with the Swedish national guidelines for patients with a low risk of infection. Better compliance to the guideline recommendation to use a urine dipstick would probably increase the number of patients classified as having an increased risk of infection. Being classified as a high-risk patient should lead to an extended duration of antibiotic prophylaxis, however, the variety of regimens used in the high-risk group reflects an inability to treat these patients in a standardized fashion and also highlights a need for more clear-cut guidelines. Pre-biopsy identification of high-risk patients is an important issue to tackle for the urologic clinics in order to reduce the number of infections.\n\n\nData availability\n\nSwedish National Data Service: Current routines for antibiotic prophylaxis prior to transrectal prostate biopsy – a national survey to all urology clinics in Sweden. https://doi.org/10.5878/zdne-z98418.\n\nSND1137-001-V1.0.zip contains the following underlying data:\n\nData_Survey_TRUS-biopsy-prophylaxis_Sweden.csv (results of the survey in CSV format).\n\nData_Survey_TRUS-biopsy-prophylaxis_Sweden.xlsx (results of the survey in Microsoft Excel format).\n\nVariable_list_TRUS-biopsy-prophylaxis_Sweden.pdf (list of variables used in the dataset).\n\nSwedish National Data Service: Current routines for antibiotic prophylaxis prior to transrectal prostate biopsy – a national survey to all urology clinics in Sweden. https://doi.org/10.5878/zdne-z98418.\n\nSND1137-001-V1.0.zip contains the following underlying data:\n\nSurvey_TRUS-biopsy-prophylaxis_Sweden.pdf (copy of the survey in English).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors thank Maria Nyberg for assisting in the collection of the surveys.\n\n\nReferences\n\nLoeb S, van den Heuvel S, Zhu X, et al.: Infectious complications and hospital admissions after prostate biopsy in a European randomized trial. Eur Urol. 2012; 61(6): 1110–4. PubMed Abstract \| Publisher Full Text\n\nLundström KJ, Drevin L, Carlsson S, et al.: Nationwide population based study of infections after transrectal ultrasound guided prostate biopsy. J Urol. 2014; 192(4): 1116–22. PubMed Abstract \| Publisher Full Text\n\nZani EL, Clark OA, Rodrigues Netto N Jr: Antibiotic prophylaxis for transrectal prostate biopsy. Cochrane Database Syst Rev. 2011; (5): CD006576. PubMed Abstract \| Publisher Full Text\n\nPu C, Bai Y, Yuan H, et al.: Reducing the risk of infection for transrectal prostate biopsy with povidone-iodine: a systematic review and meta-analysis. Int Urol Nephrol. 2014; 46(9): 1691–8. PubMed Abstract \| Publisher Full Text\n\nPilatz A, Pradere B, Yuan Y, et al.: Non-antibiotic strategies for reducing infective complications in men undergoing prostate biopsy: A systematic review and meta-analysis. Eur Urol Suppl. 2016; 15(3): e157. Publisher Full Text\n\nBorghesi M, Ahmed H, Nam R, et al.: Complications After Systematic, Random, and Image-guided Prostate Biopsy. Eur Urol. 2017; 71(3): 353–65. PubMed Abstract \| Publisher Full Text\n\nYang L, Gao L, Chen Y, et al.: Prophylactic Antibiotics in Prostate Biopsy: A Meta-Analysis Based on Randomized Controlled Trials. Surg Infect (Larchmt). 2015; 16(6): 733–47. PubMed Abstract \| Publisher Full Text\n\nBonkat G, Pickard R, Bartoletti R, et al.: EAU Guidelines on Urological Infections. 2017. European Association of Urology Web site. 2017. Reference Source\n\nLiss MA, Ehdaie B, Loeb S, et al.: AUA Guidelines. The Prevention and Treatment of the More Common Complications Related to Prostate Biopsy Updated 2016. Web site. 2016. Reference Source\n\nRegionala cancercentrum i samverkan. [Prostate cancer, national guidelines]. 2017. Reference Source\n\nTogo Y, Kubo T, Taoka R, et al.: Occurrence of infection following prostate biopsy procedures in Japan: Japanese Research Group for Urinary Tract Infection (JRGU) - a multi-center retrospective study. J Infect Chemother. 2014; 20(4): 232–7. PubMed Abstract \| Publisher Full Text\n\nWagenlehner FME, van Oostrum E, Tenke P, et al.: Infective complications after prostate biopsy: outcome of the Global Prevalence Study of Infections in Urology (GPIU) 2010 and 2011, a prospective multinational multicentre prostate biopsy study. Eur Urol. 2013; 63(3): 521–7. PubMed Abstract \| Publisher Full Text\n\nCarignan A, Roussy JF, Lapointe V, et al.: Increasing risk of infectious complications after transrectal ultrasound-guided prostate biopsies: time to reassess antimicrobial prophylaxis? Eur Urol. 2012; 62(3): 453–9. PubMed Abstract \| Publisher Full Text\n\nTeillant A, Gandra S, Barter D, et al.: Potential burden of antibiotic resistance on surgery and cancer chemotherapy antibiotic prophylaxis in the USA: a literature review and modelling study. Lancet Infect Dis. 2015; 15(12): 1429–37. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organisation (WHO): Antimicrobial resistance global report on surveillance. 2014. Reference Source\n\nThe Public Health Agency of Sweden and National Veterinary Institute. 2016 SWEDRES SVARM. 2016.\n\nCai T, Verze P, Brugnolli A, et al.: Adherence to European Association of Urology Guidelines on Prophylactic Antibiotics: An Important Step in Antimicrobial Stewardship. Eur Urol. 2016; 69(2): 276–83. PubMed Abstract \| Publisher Full Text\n\nStyrke J, Resare S, Lundström KJ, et al.: Current routines for antibiotic prophylaxis prior to transrectal prostate biopsy - a national survey to all urology clinics in Sweden. Swedish National Data Service. Dataset. http://www.doi.org/10.5878/zdne-z984\n\nTomic K, Sandin F, Wigertz A, et al.: Evaluation of data quality in the National Prostate Cancer Register of Sweden. Eur J Cancer. 2015; 51(1): 101–11. PubMed Abstract \| Publisher Full Text\n\nCarlsson S, Bratt O, Stattin P, et al.: Current routines for transrectal ultrasound-guided prostate biopsy: a web-based survey by the Swedish Urology Network. Scand J Urol Nephrol. 2012; 46(6): 405–10. PubMed Abstract \| Publisher Full Text\n\nSamarinas M, Dimitropoulos K, Zachos I, et al.: A single dose of meropenem is superior to ciprofloxacin in preventing infections after transrectal ultrasound-guided prostate biopsies in the era of quinolone resistance. World J Urol. 2016; 34(11): 1555–9. PubMed Abstract \| Publisher Full Text\n\nFahmy AM, Kotb A, Youssif TA, et al.: Fosfomycin antimicrobial prophylaxis for transrectal ultrasound-guided biopsy of the prostate: A prospective randomised study. Arab J Urol. 2016; 14(3): 228–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAtilgan D, Gençten Y, Kölükçü E, et al.: Comparison between ciprofloxacin and trimethoprim-sulfamethoxazole in antibiotic prophylaxis for transrectal prostate biopsy. Turk J Urol. 2015; 41(1): 27–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWomble PR, Dixon MW, Linsell SM, et al.: Infection related hospitalizations after prostate biopsy in a statewide quality improvement collaborative. J Urol. 2014; 191(6): 1787–92. PubMed Abstract \| Publisher Full Text\n\nYang L, Tang Z, Gao L, et al.: The augmented prophylactic antibiotic could be more efficacious in patients undergoing transrectal prostate biopsy: a systematic review and meta-analysis. Int Urol Nephrol. 2016; 48(8): 1197–1207. PubMed Abstract \| Publisher Full Text\n\nCussans A, Somani BK, Basarab A, et al.: The role of targeted prophylactic antimicrobial therapy before transrectal ultrasonography-guided prostate biopsy in reducing infection rates: a systematic review. BJU Int. 2016; 117(5): 725–31. PubMed Abstract \| Publisher Full Text\n\nRoberts MJ, Williamson DA, Hadway P, et al.: Baseline prevalence of antimicrobial resistance and subsequent infection following prostate biopsy using empirical or altered prophylaxis: A bias-adjusted meta-analysis. Int J Antimicrob Agents. 2014; 43(4): 301–9. PubMed Abstract \| Publisher Full Text\n\nLindstedt S, Lindström U, Ljunggren E, et al.: Single-dose antibiotic prophylaxis in core prostate biopsy: Impact of timing and identification of risk factors. Eur Urol. 2006; 50(4): 832–7. PubMed Abstract \| Publisher Full Text\n\nDevillé WL, Yzermans JC, van Duijn NP, et al.: The urine dipstick test useful to rule out infections. A meta-analysis of the accuracy. BMC Urol. 2004; 4: 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBruyère F, d´Arcier BF, Boutin JM, et al.: Is urine culture routinely necessary before prostate biopsy? Prostate Cancer Prostatic Dis. 2010; 13(3): 260–2. PubMed Abstract \| Publisher Full Text\n\nvan der Starre WE, van Nieuwkoop C, Paltansing S, et al.: Risk factors for fluoroquinolone-resistant Escherichia coli in adults with community-onset febrile urinary tract infection. J Antimicrob Chemother. 2011; 66(3): 650–6. PubMed Abstract \| Publisher Full Text" }	[ { "id": "59268", "date": "14 Feb 2020", "name": "Truls Bjerklund-Johansen", "expertise": [ "Reviewer Expertise Hospital acquired infections in urology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper reports that fluoroquinolone based prophylaxis is most commonly used in transrectal prostate biopsies in Sweden and that extended prophylaxis is often used if the patient has risk factors for infective complications.\nThe main message is that practice is consistent with Swedish guidelines which is of course evidence based. Although the study is well performed and the paper is well written, there are significant concerns.\nThe paper does not address that transrectal biopsy is a severely contaminated procedure. All contaminated procedures should be replaced by clean procedures whenever possible. Prostate biopsy can now be performed via the transperineal route through disinfected skin in local anesthesia as an outpatient procedure. The paper does also not address the new regulations by EMA which states that fuoroquinolones should not be used for prophylaxis. Extended prophylaxis will reduce the rates of infective complications but violates the principles of antimicrobial stewardship. Finally, urology departments should have been asked about the local resistance rates to fluoroquinolones.\nOur clinical practice should always be based on established surgical principles of hygiene. If we do that we will also comply with the principles of antimicrobial stewardship. These principles outweigh evidence from studies comparing one antibiotic with another. Having said this, there is increasing evidence that transperineal targeted as well as systematic biopsies can be performed safely without antibiotic prophylaxis.\nSwedish guidelines as well as international guidelines on prostate biopsies need to be updated.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "62141", "date": "20 Apr 2020", "name": "Shingo Yamamoto", "expertise": [ "Reviewer Expertise urinary tract infections" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis report is important to know the real world data in terms of prophylaxis for TRUS in Sweden. However, several points are to be discussed as below.\nInfection following TRUS is not only UTI but mainly prostatitis.\n\nThus, by previous papers and guidelines, risk factors are defined as antimicrobial-resistant bacterial flora in the rectum as as well as bacteriuria in the bladder.\n\nRecently several papers reported that the culture of rectal swab is useful to prescribe prophylaxis for TRUS.\n\nSome papers recommend parenteral antimicrobials, especially in high risk patients.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-58
https://f1000research.com/articles/9-56/v1	28 Jan 20	{ "type": "Research Article", "title": "Dyslipidemia as a risk factor for liver fibrosis progression in a multicentric population with non-alcoholic steatohepatitis", "authors": [ "Nahum Méndez-Sánchez", "Eira Cerda-Reyes", "Fátima Higuera-de-la-Tijera", "Ana K. Salas-García", "Samantha Cabrera-Palma", "Guillermo Cabrera-Álvarez", "Carlos Cortez-Hernández", "Luis A Pérez-Arredondo", "Emma Purón-González", "Edgar Coronado-Alejandro", "Arturo Panduro", "Heriberto Rodríguez-Hernández", "Vania C. Cruz-Ramón", "Alejandro Valencia-Rodríguez", "Xingshun Qi", "Nashla Hamdan-Pérez", "Nancy E. Aguilar-Olivos", "Beatriz Barranco-Fragoso", "Oscar Ramírez-Pérez", "Alfonso Vera-Barajas", "Eira Cerda-Reyes", "Fátima Higuera-de-la-Tijera", "Ana K. Salas-García", "Samantha Cabrera-Palma", "Guillermo Cabrera-Álvarez", "Carlos Cortez-Hernández", "Luis A Pérez-Arredondo", "Emma Purón-González", "Edgar Coronado-Alejandro", "Arturo Panduro", "Heriberto Rodríguez-Hernández", "Vania C. Cruz-Ramón", "Alejandro Valencia-Rodríguez", "Xingshun Qi", "Nashla Hamdan-Pérez", "Nancy E. Aguilar-Olivos", "Beatriz Barranco-Fragoso", "Oscar Ramírez-Pérez", "Alfonso Vera-Barajas" ], "abstract": "Background: Nonalcoholic fatty liver disease (NAFLD) is a serious worldwide health problem, with an estimated global prevalence of 24%; it has a notable relationship with other metabolic disorders, like obesity and type 2 diabetes mellitus (T2DM). Nonalcoholic steatohepatitis (NASH) is one of the most important clinical entities of NAFLD, which is associated with an increased risk of progression to liver cirrhosis and hepatocellular carcinoma (HCC). Mexico is one of the countries with the highest prevalence of metabolic diseases; therefore, we sought to investigate the impact that these clinical entities have in the progression to advanced fibrosis in Mexican patients with NASH. Methods: We performed a multicenter retrospective cross-sectional study, from January 2012 to December 2017. A total of 215 patients with biopsy-proven NASH and fibrosis were enrolled. NASH was diagnosed according NAS score and liver fibrosis was staged by the Kleiner scoring system. For comparing the risk of liver fibrosis progression, we divided our sample into two groups. Those patients with stage F0-F2 liver fibrosis were included in the group with non-significant liver fibrosis (n=178) and those individuals with F3-F4 fibrosis were included in the significant fibrosis group (n=37). We carried out a multivariate analysis to find risk factors associated with liver fibrosis progression. Results: From the 215 patients included, 37 had significant liver fibrosis (F3-4). After logistic regression analysis T2DM (p=0.044), systemic arterial hypertension (p=0.014), cholesterol (p=0.041) and triglycerides (p=0.015) were the main predictor of advanced liver fibrosis. Conclusions: In a Mexican population, dyslipidemia was the most important risk factor associated with advanced liver fibrosis and cirrhosis.", "keywords": [ "non-alcoholic fatty liver disease", "cirrhosis", "dyslipidemia", "type 2 diabetes mellitus", "metabolic syndrome." ], "content": "Introduction\n\nNonalcoholic fatty liver disease (NAFLD) has a broad clinical spectrum, ranging from simple steatosis to cirrhosis, even developing in some cases with hepatocellular carcinoma (HCC)1. Nonalcoholic steatohepatitis (NASH) is one of the most important clinical entities of NAFLD, characterized by the histologic presence of liver steatosis, ballooning degeneration, and lobular inflammation, with or without fibrosis2. Once NASH is established, there is a significant increased risk of developing liver cirrhosis and HCC3.\n\nCurrently, NAFLD is the most common chronic liver disease in the world, with an important relationship with other metabolic disorders like obesity, type 2 diabetes mellitus (T2DM) and metabolic syndrome (MetS)4–6, being the second leading indication for liver transplantation in the United States7. A global prevalence of 24% is estimated, with the highest rates in South America and the Middle East, while the lowest prevalence has been reported in Africa8.\n\nIt is estimated that 30–40% of NAFLD patients will develop NASH9,10. Some studies have demonstrated that the risk of progression to liver cirrhosis in NAFLD patients is between 0–4%, while approximately 10–25% of NASH patients will develop cirrhosis11–16. This also depends on the ethnic origin of the patients, since Hispanic-Americans have been found to have a wide susceptibility to NAFLD and NASH development mainly from Mexican origin (33%)8.\n\nMultiple risk factors for NASH progression have been identified, such as diet, MetS, T2DM, obesity, Hispanic ethnicity and polymorphisms in the patatin-like phospholipase domain-containing 3 (PNPLA3) gene17–19. However, the pathological mechanisms, by which some NAFLD patients progress to NASH are still not well understood20.\n\nMexico is one of the countries with the highest prevalence of metabolic diseases; 75.2% of the Mexican population are obese or overweight, 10.3% have T2DM and 19.5% have dyslipidemia21. We therefore sought to investigate the main metabolic factors involved in the progression to advanced fibrosis in Mexican patients with NASH.\n\n\nMethods\n\nWe conducted a multicenter retrospective cross-sectional study from January 2012 to December 2017 in 7 tertiary referral centers from different parts of Mexico: Medica Sur Clinic and Foundation (Mexico City), General Hospital of Mexico “Dr. Eduardo Liceaga” (Mexico City), Civil Hospital of Guadalajara “Fray Antonio Alcalde” (Jalisco), Christus Muguerza “Super Speciality” Hospital (Nuevo Leon), Central Military Hospital (Mexico City), General Hospital of the Mexican Social Security Institute (Durango), and the General Regional Hospital, IMSS 1 (Morelos).\n\nThis study was reviewed and approved by the Ethics Committee of the Medica Sur Clinic and Foundation. Patients were not required to give informed consent to the study because the analysis used anonymous clinical data.\n\nWe included patients older than 20 years, of both genders, who had the diagnosis of biopsy-proven NASH. NASH was diagnosed according to NAS score5,6, and liver fibrosis was staged by the Kleinier scoring system15. We reviewed the medical records to obtain the baseline characteristics, biochemical parameters and comorbidities excluding all patients with other causes of liver disease, such as alcoholic liver disease, hepatitis B virus, hepatitis C virus, autoimmune liver disease, iron overload, drug-induced liver disease, and hereditary liver disease.\n\nSeveral variables that have been commonly associated as risk factors for NASH progression were studied and included in the statistical analyses, such as MetS diagnosed with three or more of the following criteria established by the National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III)22: waist circumference over 102 cm (men) or 88 cm (women), blood pressure over 130/85 mmHg, fasting triglyceride (TG) level over 150 mg/dl, fasting high-density lipoprotein (HDL) cholesterol level less than 40 mg/dl (men) or 50 mg/dl (women) and fasting blood glucose over 100 mg/dl. To define overweight or obesity we use the body mass index (BMI) defined as weight (in kg) / height2 (in m2). Where overweight is defined above 25 kg / m2, and obesity as > 30 kg / m2. T2DM was diagnosed according to the history of diseases, fasting glucose (>126 mg/dL), and/or glucose tolerance test (>200 mg/dL two hours after the glucose load). Systemic arterial hypertension was diagnosed according to the history of diseases and/or intake of antihypertensive drugs. Cardiovascular disease (CVD) was defined as the presence of history such as systemic arterial hypertension, coronary heart disease, previous heart attack or stroke. Laboratory evaluation in all patients included serum liver tests (aspartate transaminase, alanine transaminase, total bilirubin, alkaline phosphatase, and albumin), hemoglobin, platelet count, cholesterol, low-density lipoprotein, HDL, TG, and blood glucose. The diagnosis of HCC was based on histopathological findings by liver biopsy.\n\nFor comparing the risk of liver fibrosis progression, we divided our sample into two groups. Those patients with stage F0-F2 liver fibrosis were included in the group with non-significant liver fibrosis (n=178) and the individuals with stage F3-F4 fibrosis were included in the significant fibrosis group (n=37).\n\nWe produced descriptive statistics for baseline patient characteristics. Univariate analysis of quantitative variables was conducted using Student’s t-test, while for qualitative variables we used the Chi square test. Multivariate regression analyses were used to assess the independent association between significant fibrosis and possible risk factors. A p-value <0.05 was determined to indicate significance. All analyses were performed using STATA (v14.1, Stata Corp, College Station, TX, USA).\n\n\nResults\n\nWe included 215 patients (Table 1). Non-significant fibrosis (F0-F2) was found in 83% of our sample; 7% had fibrosis at stage F-3, and 10% had fibrosis at stage F-4. Five patients were diagnosed with HCC (Table 2). De-identified individual-level results are available as Underlying data23.\n\nCompared to the group with non-significant liver fibrosis (stages F0, F1 and F2), those with significant fibrosis (stages F3 or F4) were more likely to be older and have lower levels of platelet count and albumin (Table 3 and Table 4). Moreover, those with significant fibrosis showed higher levels of blood glucose and proportions of dyslipidemia, T2DM, MetS, systemic arterial hypertension, and CVD.\n\nLogistic multivariate regression analysis demonstrated that T2DM, systemic arterial hypertension, high cholesterol and hypertriglyceridemia significantly increased the risk for advanced liver fibrosis (Table 5), but only T2DM and hypertriglyceridemia significantly raised the risk of developing cirrhosis. (Table 6).\n\n\nDiscussion\n\nIn this study we found that T2DM, triglycerides and cholesterol were the main factors associated with advanced fibrosis stages and cirrhosis. In this line, the association between T2DM and NASH progression has been widely demonstrated in a large number of studies from different geographical points24–28. Our findings support this association in patients of Mexican origin. On the other hand, in recent years some experimental and clinical studies have suggested an important role of lipid deregulation in the progression of fibrosis. Even though hepatic accumulation of triglycerides is the main feature of NAFLD, it has only been related with the development of simple steatosis due to its low lipotoxic characteristics. Contrarily, free cholesterol (FC) is an important lipotoxic species commonly found in NASH patients that has been linked to hepatic inflammation and fibrosis29.\n\nThe first cholesterol-induced NASH hypothesis was proposed more than 10 years ago by suggesting that mitochondrial FC loading changed the fluidity of the mitochondrial membranes, leading to the oxidation of mitochondrial glutathione, and sensitized hepatocytes to tumor necrosis factor and Fas-dependent death signaling via mitochondrial glutathione depletion, exacerbating NASH30. Furthermore, hyperinsulinemia (another characteristic usually found in NASH) and cholesterol accumulation was seen to upregulate sterol regulatory element-binding protein 2 (SREBP-2), leading to increased cholesterol synthesis and downregulation of mitochondrial β-oxidation, with the consequent accumulation of cholesterol and free fatty acids in the liver31. All these events precipitate hepatocyte injury, apoptosis, macrophage recruitment, liver fibrosis, and progression from steatosis to NASH32.\n\nMore recent evidence has found that when feeding C57BL/6 mice with a high cholesterol + methionine/choline-deficient diet for 12 weeks, there was an increase in the development of fibrosis due to the intrahepatic accumulation of FC in the hepatic stellate cells (HSCs) via TLR4 activation with the consequent downregulation of the transforming growth factor-beta (TGF-β) pseudoreceptor bone morphogenetic protein and activin membrane-bound inhibitor, leading to TGF-β-induced fibrosis33.\n\nIn addition, FC was found to alter the SREBP2-mediated feedback system, likely due to an increase in the expression of the SREBP cleavage-activating protein (SCAP) and the lack of expression of insulin induced gene 2 (Insig-2) in HSCs which favored the accumulation of cholesterol and the transdifferentiation of HSCs into myofibroblasts33.\n\nIn humans, a retrospective study conducted in cirrhotic patients found that cholesterol was a strong mortality predictor in those patients34. Also, in a more recent study, a group of NAFLD surgical specimens was studied observing an accumulation of FC and oxidized low-density lipoprotein in the portal vein and the hepatic sinusoidal parenchyma in the form of cholesterol crystals, related to the activation of NOD-like receptor family pyrin domain containing protein 3 (NLRP3) inflammasome promoting inflammation, HSCs activation and a characteristic chicken-wire fibrosis35. Furthermore, basic research has demonstrated that cholesterol crystals (lipid droplets with a strong birefringence under polarized light) are only present in NASH-models (not in simple steatosis). Under these conditions, activated Kupffer cells (KCs) aggregate along cholesterol crystals forming “crown-like structures” intimately related to the development of foam cells and therefore atherosclerosis36.\n\nThe latter demonstrates the importance of CVD with NASH, being the first cause of death in this group of patients. Therefore, our findings demonstrate the important relation that metabolic diseases have with advanced stages of NASH. This will lead to the necessity of better preventive programs with a greater impact and coverage for general population around the world.\n\nFinally, we must point out the limitations of this study such as the cross-sectional design which precluded the establishment of causal and temporal relationships between NASH progression and associated variables. Also, the small number of patients with significant fibrosis was important to mention.\n\nIn conclusion, our results show that dyslipidemia is one of the main risk factors associated with the development of significant liver fibrosis stages. Hispanics/Latinos and the Mexican population have one of the highest rates of metabolic diseases around the world; for this reason, it is expected that NAFLD and NASH will be the leading cause of cirrhosis in the near future. Therefore, it could be crucial to develop better screening programs for NAFLD patients with hypercholesterolemia and/or another component of MetS in order to prevent the development of more significant clinical complications.\n\n\nData availability\n\nHarvard Dataverse: Dyslipidemia as a risk factor for liver fibrosis progression in a multicentric population with non-alcoholic steatohepatitis. https://doi.org/10.7910/DVN/EE3DIC23.\n\nThis project contains de-identified data for each patient assessed in this study.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nMéndez-Sánchez N, Arrese M, Zamora-Valdés D, et al.: Current concepts in the pathogenesis of nonalcoholic fatty liver disease. Liver Int. 2007; 27(4): 423–33. PubMed Abstract \| Publisher Full Text\n\nMachado MV, Diehl AM: Pathogenesis of Nonalcoholic Steatohepatitis. Gastroenterology. 2016; 150(8): 1769–77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrunt EM: Nonalcoholic steatohepatitis: definition and pathology. Semin Liver Dis. 2001; 21(1): 3–16. PubMed Abstract \| Publisher Full Text\n\nOng JP, Younossi ZM: Epidemiology and natural history of NAFLD and NASH. Clin Liver Dis. 2007; 11(1): 1–16, vii. PubMed Abstract \| Publisher Full Text\n\nMéndez-Sánchez N, Chavez-Tapia NC, Almeda-Valdes P, et al.: The management of incidental fatty liver found on imaging. What do we need to do? Am J Gastroenterol. 2018; 113(9): 1274–6. PubMed Abstract \| Publisher Full Text\n\nYounossi Z, Anstee QM, Marietti M, et al.: Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol. 2018; 15(1): 11–20. PubMed Abstract \| Publisher Full Text\n\nChedid MF: Nonalcoholic Steatohepatitis: The Second Leading Indication for Liver Transplantation in the USA. Dig Dis Sci. 2017; 62(10): 2621–2. PubMed Abstract \| Publisher Full Text\n\nYounossi ZM, Koenig AB, Abdelatif D, et al.: Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology. 2016; 64(1): 73–84. PubMed Abstract \| Publisher Full Text\n\nPerumpail BJ, Khan MA, Yoo ER, et al.: Clinical epidemiology and disease burden of nonalcoholic fatty liver disease. World J Gastroenterol. 2017; 23(47): 8263–76. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCalzadilla-Bertot L, Adams LA: The Natural Course of Non-Alcoholic Fatty Liver Disease. Int J Mol Sci. 2016; 17(5): pii: E774. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTeli MR, James OF, Burt AD, et al.: The natural history of nonalcoholic fatty liver: a follow-up study. Hepatology. 1995; 22(6): 1714–9. PubMed Abstract \| Publisher Full Text\n\nEkstedt M, Franzén LE, Mathiesen UL, et al.: Long-term follow-up of patients with NAFLD and elevated liver enzymes. Hepatology. 2006; 44(4): 865–73. PubMed Abstract \| Publisher Full Text\n\nLoomba R, Abraham M, Unalp A, et al.: Association between diabetes, family history of diabetes, and risk of nonalcoholic steatohepatitis and fibrosis. Hepatology. 2012; 56(3): 943–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDe Minicis S, Day C, Svegliati-Baroni G: From NAFLD to NASH and HCC: pathogenetic mechanisms and therapeutic insights. Curr Pharm Des. 2013; 19(29): 5239–49. PubMed Abstract \| Publisher Full Text\n\nKleiner DE, Brunt EM, Van Natta M, et al.: Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005; 41(6): 1313–21. PubMed Abstract \| Publisher Full Text\n\nDongiovanni P, Rametta R, Meroni M, et al.: The role of insulin resistance in nonalcoholic steatohepatitis and liver disease development--a potential therapeutic target? Expert Rev Gastroenterol Hepatol. 2016; 10(2): 229–42. PubMed Abstract \| Publisher Full Text\n\nYang KC, Hung HF, Lu CW, et al.: Association of Non-alcoholic Fatty Liver Disease with Metabolic Syndrome Independently of Central Obesity and Insulin Resistance. Sci Rep. 2016; 6: 27034. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKakisaka K, Suzuki Y, Fujiwara Y, et al.: Evaluation of ballooned hepatocytes as a risk factor for future progression of fibrosis in patients with non-alcoholic fatty liver disease. J Gastroenterol. 2018; 53(12): 1285–91. PubMed Abstract \| Publisher Full Text\n\nChinchilla-López P, Ramírez-Pérez O, Cruz-Ramón V, et al.: More Evidence for the Genetic Susceptibility of Mexican Population to Nonalcoholic Fatty Liver Disease through PNPLA3. Ann Hepatol. 2018; 17(2): 250–5. PubMed Abstract \| Publisher Full Text\n\nBenedict M, Zhang X: Non-alcoholic fatty liver disease: An expanded review. World J Hepatol. 2017; 9(16): 715–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRomero-Martínez M, Shamah-Levy T, Vielma-Orozco E, et al.: [National Health and Nutrition Survey 2018-19: methodology and perspectives]. Salud Publica Mex. 2019; 61(6): 917–23. PubMed Abstract \| Publisher Full Text\n\nNational Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III): Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) final report. Circulation. 2002; 106(25): 3143–421. PubMed Abstract \| Publisher Full Text\n\nMéndez-Sánchez N, Valencia-Rodríguez A, Higuera-de-la-Tijera F, et al.: \"Dyslipidemia as a risk factor for liver fibrosis progression in a multicentric population with non-alcoholic steatohepatitis\". Harvard Dataverse, V1, 2020. http://www.doi.org/10.7910/DVN/EE3DIC\n\nAngulo P, Keach JC, Batts KP, et al.: Independent predictors of liver fibrosis in patients with nonalcoholic steatohepatitis. Hepatology. 1999; 30(6): 1356–62. PubMed Abstract \| Publisher Full Text\n\nHossain N, Afendy A, Stepanova M, et al.: Independent predictors of fibrosis in patients with nonalcoholic fatty liver disease. Clin Gastroenterol Hepatol. 2009; 7(11): 1224–1229. PubMed Abstract \| Publisher Full Text\n\nPuchakayala BK, Verma S, Kanwar P, et al.: Histopathological differences utilizing the nonalcoholic fatty liver disease activity score criteria in diabetic (type 2 diabetes mellitus) and non-diabetic patients with nonalcoholic fatty liver disease. World J Hepatol. 2015; 7(25): 2610–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNakahara T, Hyogo H, Yoneda M, et al.: Type 2 diabetes mellitus is associated with the fibrosis severity in patients with nonalcoholic fatty liver disease in a large retrospective cohort of Japanese patients. J Gastroenterol. 2014; 49(11): 1477–84. PubMed Abstract \| Publisher Full Text\n\nPorepa L, Ray JG, Sanchez-Romeu P, et al.: Newly diagnosed diabetes mellitus as a risk factor for serious liver disease. CMAJ. 2010; 182(11): E526–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIoannou GN: The Role of Cholesterol in the Pathogenesis of NASH. Trends Endocrinol Metab. 2016; 27(2): 84–95. PubMed Abstract \| Publisher Full Text\n\nMarí M, Caballero F, Colell A, et al.: Mitochondrial free cholesterol loading sensitizes to TNF- and Fas-mediated steatohepatitis. Cell Metab. 2006; 4(3): 185–98. PubMed Abstract \| Publisher Full Text\n\nVan Rooyen DM, Farrell GC: SREBP-2: a link between insulin resistance, hepatic cholesterol, and inflammation in NASH. J Gastroenterol Hepatol. 2011; 26(5): 789–92. PubMed Abstract \| Publisher Full Text\n\nTirosh O: Hypoxic Signaling and Cholesterol Lipotoxicity in Fatty Liver Disease Progression. Oxid Med Cell Longev. 2018; 2018: 2548154. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTomita K, Teratani T, Suzuki T, et al.: Free cholesterol accumulation in hepatic stellate cells: mechanism of liver fibrosis aggravation in nonalcoholic steatohepatitis in mice. Hepatology. 2014; 59(1): 154–69. PubMed Abstract \| Publisher Full Text\n\nJaničko M, Veselíny E, Leško D, et al.: Serum cholesterol is a significant and independent mortality predictor in liver cirrhosis patients. Ann Hepatol. 2013; 12(4): 581–7. PubMed Abstract \| Publisher Full Text\n\nHo CM, Ho SL, Jeng YM, et al.: Accumulation of free cholesterol and oxidized low-density lipoprotein is associated with portal inflammation and fibrosis in nonalcoholic fatty liver disease. J Inflamm (Lond). 2019; 16(1): 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIoannou GN, Haigh WG, Thorning D, et al.: Hepatic cholesterol crystals and crown-like structures distinguish NASH from simple steatosis. J Lipid Res. 2013; 54(5): 1326–34. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "59237", "date": "18 Feb 2020", "name": "Kevork Peltekian", "expertise": [ "Reviewer Expertise Hepatology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors should be congratulated for prospectively collecting information regarding risk factors for advanced liver fibrosis in patients with non-alcoholic steatohepatitis. The medical literature lacks enough data. The main predictors obviously include thrombocytopenia and components of metabolic syndrome.\n\nThe authors may want to discuss the metabolic syndrome is an independent factor for predicting more advanced fibrosis (if the statistics confirms it).\nThe discussion can be simplified by minimizing results of animal studies and enhancing more results of human studies.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5245", "date": "19 Feb 2020", "name": "Nahum Méndez-Sánchez", "role": "Author Response", "response": "Dear Dr. Peltekian, We appreciate your comments and we are honored to have had the opportunity of being evaluated by a distinguished member of the hepatology field. We agree that not all animal studies are reproducible in humans, however, we believe that to support our results it is important to include them since currently there is an important lack of information in humans about dyslipidemia as a risk factor for liver fibrosis progression." } ] }, { "id": "59240", "date": "16 Jun 2020", "name": "Piero Portincasa", "expertise": [ "Reviewer Expertise Internal medicine", "metabolic disorders", "lipidology", "hepatology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a multicentre retrospective cross-sectional study on NAFLD (2012-17) with 215 patients undergoing liver histology (NASH-fibrosis). Group F0-F2 fibrosis and Group F3-F4 were compare by logistic regression analysis. T2DM, systemic arterial hypertension, cholesterol and triglycerides were the main predictor of advanced liver fibrosis\nThe paper is well presented and simple in the take-home message. The authors conclude that T2DM, serum triglycerides and cholesterol were the main factors associated with advanced fibrosis stages and cirrhosis.\n\nA general point is to discuss the limited n. of patients with fibrosis (N=37) and how this number could affect the overall results of the study. This aspect is only briefly touched at the end of discussion.\n\nAlso, can the authors provide any information about pre-T2DM, i.e. the presence of insulin resistance?\n\nAbstract: please better describe the terms “cholesterol” and “triglycerides” (serum chol, TG)\n\nIntro: the authors should also mention the recent consensus paper in gastro re-classifying the metabolic NAFLD Eslam M, Sanyal AJ, George J, International Consensus Panel. MAFLD: A Consensus-Driven Proposed Nomenclature for Metabolic Associated Fatty Liver Disease1.\n\nWhen mentioning PNPLA3, please refer to a previous paper Krawczyk M, Portincasa P, Lammert F. PNPLA3-associated steatohepatitis: toward a gene-based classification of fatty liver disease2.\n\nTable 1: The table should bring the total number of enrolled patients at the top. I would suggest to simplify by mentioning at the bottom: data are mean (SD)\n\nDiscussion: The study is only observational and not longitudinal (till 2017). Can the authors provide info about the follow-up of some of these patients with advanced fibrosis?\n\nI also would spend few words about the role of therapeutic agents (hypolipidemic drugs) on limiting the progression of NASH\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-56
https://f1000research.com/articles/9-50/v1	27 Jan 20	{ "type": "Case Report", "title": "Case Report: Isolated hepatosplenic sarcoidosis treatment improving glycaemic control in a type 1 diabetic patient", "authors": [ "Sérgio Pina", "Teresa Salero", "Mariana Figueiras", "Rui Osório", "Sofia Amálio", "Teresa Salero", "Mariana Figueiras", "Rui Osório", "Sofia Amálio" ], "abstract": "Sarcoidosis is a multi-systemic disease characterized by non-caseating granulomas in various organs. The aetiology is still unknown. Although the liver is the third most commonly affected organ, hepatosplenic sarcoidosis without lung involvement is very uncommon. There is a high frequency of certain autoimmune illnesses observed in sarcoidosis, but association with type 1 diabetes is infrequent. We present the case of a 31-year-old woman, with type 1 diabetes mellitus diagnosed 22 years before with a glycated haemoglobin (HbA1c) above 14%, diabetic nephropathy, retinopathy and neuropathy, hypercholesterolemia and beta thalassemia. She was medicated with an angiotensin-converting enzyme inhibitor, a dihydropyridine calcium antagonist and insulin.\n\nThe patient presented with a 4-month history of tiredness, abdominal pain, weight lost and hepatosplenomegaly. Abdominal ultrasound revealed hepatomegaly with regular contours, diffuse heterogeneous texture, containing numerous nodules with slight enlargement of the spleen. Serum angiotensin converting enzyme (ACE) was 67 IU/L and a sedimentation rate of 120 mm/h. Computer tomography (CT) scan confirmed hepatosplenomegaly and suggested infiltration in both organs. Liver biopsies were compatible with sarcoidosis. After ruling other organ involvement, a diagnosis of isolated hepatosplenic sarcoidosis was provided and prednisolone (40mg/day) was started. After a few months the patient developed a corticoid-induced myopathy confirmed with electromyography. Prednisolone was reduced to 20mg/day and azathioprine (50mg/day) treatment initiated. After a 7-month treatment, chest-abdomen-pelvis CT scan showed a marked reduction of the nodularity and hepatosplenomegaly and after 1 year the patient was completely asymptomatic (HbA1c, 7.5%; ACE, 24IU/L). At 18-month follow-up there was no evidence of recurrence (HbA1c, 7%), with optimum glycaemic control with total daily insulin dose lowered to half. This is an uncommon case in which the treatment of hepatosplenic sarcoidosis with regression of sarcoid tissue can help explain the improvement of glycaemic control in this patient.", "keywords": [ "Hepatic sarcoidosis", "Esplenic sarcoidosis", "Type 1 Diabetes", "Hepatosplenomegaly" ], "content": "Introduction\n\nSarcoidosis is a chronic multi-system pathology characterized by epithelioid granulomas without caseation or necrosis1. The highest incidence occurs between individuals aged between 25 and 40 years old and the reported prevalence varies from 20 cases per 100000 in the UK to 64 cases per 100000 in Scandinavian and African-American populations. Sarcoidosis is more frequent in women2,3, with lung and mediastinal lymph node involvement in 90% of cases. Although the liver is the third most commonly affected organ, hepatosplenic sarcoidosis without lung involvement is very uncommon4. Coexistence of sarcoidosis and immune-mediated diseases has been described in a previous case series and an association between diabetes and sarcoidosis was found, but this is rarely reported5.\n\nWe report an uncommon case of glycaemic control in a type 1 diabetic after successful isolated hepatosplenic sarcoidosis treatment with immunosuppressant treatment.\n\n\nCase presentation\n\nA 31-year-old woman, with type 1 diabetes mellitus, diagnosed 22 years before, hypercholesterolemia and beta thalassemia. The patient had history of a poor metabolic control with glycated haemoglobin (HbA1c) above 14% (normal range 4–5.6%), diabetic nephropathy, retinopathy and neuropathy and arterial hypertension. She was medicated with 10 mg ramipril per day, 5 mg amlodipine per day, insulin detemir 26 IU in the morning and 20 IU at night, as well as prandial aspart insulin determined by pre-meal glucose level, meal size and content. The patient was allergic to glargine insulin.\n\nThe patient presented at a diabetology consult in July 2016 with a 4-month history of tiredness, anorexia, abdominal pain and weight loss (8% of total weight). At physical examination she was found to have hepatosplenomegaly. The smooth, regular liver edge was felt 4 cm below the right costal margin for a total span of 14 cm. An urgent abdominal ultrasound was performed revealing hepatomegaly with regular contours, diffuse heterogeneous texture, containing numerous hyperechogenic, nodular, confluent, mostly infracentimetric images, the largest reaching about 17 mm in diameter. The spleen was slightly enlarged with no other significant alterations.\n\nOn August 2016, the patient was admitted to the Internal Medicine ward, Hospital de Faro, for further investigation. On admittance, the patient presented no other alterations at physical exam, besides those described above related to the abdomen.\n\nBlood tests were performed, including blood count and blood cultures, electrolytes, hepatic viruses, autoantibodies, C3 and C4 complement levels, immunoglobulins, serum protein electrophoresis, sedimentation rate and serology for multiple granulomatous diseases (results in Table 1). Laboratory results of note were elevated serum angiotensin converting enzyme (ACE) of 67 IU/L, a sedimentation rate of 120 mm/h, a gamma polyclonal peak in protein electrophoresis, a hepatitis C viral (HCV) titre of 90 IU/mL and doubtful HCV antibody reaction with negative viral load. Asymptomatic hypercalcemia was also detected that was promptly corrected with isotonic saline hydration and 4mg of zoledronic acid intravenously over 15 min. Hypochromic microcytic anaemia was due to thalassemia history.\n\nFurther imaging studies were performed. Chest x-ray revealed no important changes (Figure 1), but chest-abdomen-pelvis computer tomography (CT) scan confirmed hepatosplenomegaly and revealed infiltration in both organs. No other alterations were found (Figure 2).\n\nBone marrow biopsy showed sideropenic bone marrow with reactive histological characteristics. To confirm the aetiology, liver biopsies were performed, which revealed granulomatous inflammation, non-caseating granulomas, with no necrosis, acid-fast bacilli, fungi or other organisms (Figure 3).\n\nBased on the above findings, a diagnosis of sarcoidosis was strongly favoured, and a diagnosis of isolated hepatosplenic sarcoidosis was confirmed after ruling out skin, ganglionar and ophthalmic involvement.\n\nThe patient was discharged to outpatient consultation and medicated with prednisolone (40 mg per day) after testing negative for latent tuberculosis. Following a few months of treatment, she presented with fatigue, pelvic girdle muscle weakness and muscle pain. Corticosteroid-induced myopathy was diagnosed (later confirmed with electromyography) and prednisolone was reduced to 20 mg per day, and azathioprine was added (50 mg per day) for maintenance.\n\nAfter 7 months of treatment, chest-abdomen-pelvis CT scan showed a marked reduction of the nodularity and hepatosplenomegaly (Figure 4). Other tests supported the new imaging results, such as reduction of sedimentation rate to 56 mm/h. Surprisingly, there was a progressive improvement of HbA1c to 9.5%. HCV serologies came back negative, suggesting cross reaction.\n\nAfter one year of treatment, the patient was completely asymptomatic and insulin needs had diminished. HbA1c continued to drop to 7.5% and ACE was 24 IU/L. At 18-month follow-up there was no evidence of recurrence, HbA1c was 7%, with optimum glycaemic control with total daily insulin dose lowered to half.\n\n\nDiscussion\n\nSarcoidosis is a disease of unknown aetiology that can implicate almost any organ, but most commonly affects the lung, the lymph nodes, eye and skin1,6. Involvement of the gastrointestinal tract is infrequent and hepatic sarcoidosis without lung disease is documented in only 13% of patients with systemic sarcoidosis6. It can be very challenging to diagnose since liver and spleen involvement are usually asymptomatic and functional derangement is not common. If not totally asymptomatic, the clinical presentation of hepatosplenic sarcoidosis can be weakness, weight loss and hepatosplenomegaly6,7. Our patient presented with non-specific systemic symptoms, such as poor glycaemic control, tiredness, anorexia, abdominal pain and enlargement of liver and spleen. The diagnosis was confirmed using CT scan imaging and liver biopsy.\n\nSarcoidosis dysregulates vitamin D production, increasing extrarenal production by macrophages in granulomas resulting in elevated levels of 1,25-dihydroxyvitamin D8, that can help explain the asymptomatic hypercalcemia in this case.\n\nThe liver plays a central role in the control of glucose metabolism, especially in diabetic patients, by controlling various pathways, including glycogenesis, glycogenolysis, glycolysis, gluconeogenesis and helping with insulin sensitivity9,10.\n\nThe regression of hepatic sarcoid tissue after immunosuppressant treatment restored some of the capacity of the patient’s liver to play its central role in glucose metabolism leading to a marked reduction in her insulin needs and in HbA1c with better metabolic control.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for the publication of the report and any associated images.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nBargagli E, Prasse A: Sarcoidosis: a review for the internist. Intern Emerg Med. 2018; 13(3): 325–331. PubMed Abstract \| Publisher Full Text\n\nBakker GJ, Haan YCL, Maillette de Buy Wenniger LJ, et al.: Sarcoidosis of the liver: to treat or not to treat? Neth J Med. 2012; 70(8): 349–56. PubMed Abstract\n\nRybicki BA, Major M, Popovich J Jr, et al.: Racial Differences in Sarcoidosis Incidence: A 5-Year Study in a Health Maintenance Organization. Am J Epidemiol. 1997; 145(3): 234–41. PubMed Abstract \| Publisher Full Text\n\nTadros M, Forouhar F, Wu GY: Review Article Hepatic Sarcoidosis. 2013; 1: 87–93.\n\nPietinalho A, Hiraga Y, Hosoda Y, et al.: The frequency of sarcoidosis in Finland and Hokkaido, Japan. A comparative epidemiological study. Sarcoidosis. 1995; 12(1): 61–7. PubMed Abstract\n\nHarder H, Buchler MW, Fröhlich B, et al.: Extrapulmonary sarcoidosis of liver and pancreas: a case report and review of literature. World J Gastroenterol. 2007; 13(17): 2504–2509. PubMed Abstract \| Free Full Text\n\nMueller S, Boehme MW, Hofmann WJ, et al.: Extrapulmonary sarcoidosis primarily diagnosed in the liver. Scand J Gastroenterol. 2000; 35(9): 1003–1008. PubMed Abstract \| Publisher Full Text\n\nBaughman RP, Papanikolaou I: Current concepts regarding calcium metabolism and bone health in sarcoidosis. Curr Opin Pulm Med. 2017; 23(5): 476–481. PubMed Abstract \| Publisher Full Text\n\nKumar R: Hepatogenous Diabetes: An Underestimated Problem of Liver Cirrhosis. Indian J Endocrinol Metab. 2018; 22(4): 552–559. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHan HS, Kang G, Kim JS, et al.: Regulation of glucose metabolism from a liver-centric perspective. Exp Mol Med. 2016; 48(3): e218. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "60097", "date": "18 Feb 2020", "name": "Wilbert S Aronow", "expertise": [ "Reviewer Expertise cardiovascular disease" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an excellent case report which shows that treatment of hepatosplenic sarcoidosis with regression of sarcoid tissue can improve glycemic control in a diabetic treated with insulin.\n\nCoexistence of sarcoidosis and immune-mediated diseases has been described in a previous case series and an association between diabetes and sarcoidosis was found, but this is rarely reported.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "68765", "date": "10 Aug 2020", "name": "Sara Ghoneim", "expertise": [ "Reviewer Expertise Hepatology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDiscuss any relationships you might have found between the association of T1DM and sarcoidosis.\n\nPlease change Tiredness to fatigue or lethargy whatever you see is more appropriate.\n\nThe discussion needs to focus more on the pathophysiology of the disease, and the interplay between diabetes and sarcoidosis.1\n\nPlease include an \"in conclusion\" part of the discussion to summarize the case report.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] } ]	1	https://f1000research.com/articles/9-50
https://f1000research.com/articles/9-48/v1	27 Jan 20	{ "type": "Systematic Review", "title": "Efficacy and safety of microbiota transfer therapy for the management of autism spectrum disorder in children: a systematic review", "authors": [ "Pablo Daniel Estrella Porter", "Luis Eduardo Guzmán Freire", "Joseth Paulina Adatty Molina", "María Verónica Burneo Raza", "Henry Alejandro Carrión Celi", "Isabel María Espinosa Borja", "Andrea Carolina Falconí Páez", "Andrés Sebastián Gudiño Vega", "María José Jaramillo Cartwright", "Sebastián Xavier Oña Vargas", "Sebastián Eduardo Puga Martínez", "Jonathan R Guillemot", "Pablo Daniel Estrella Porter", "Luis Eduardo Guzmán Freire", "Joseth Paulina Adatty Molina", "María Verónica Burneo Raza", "Henry Alejandro Carrión Celi", "Isabel María Espinosa Borja", "Andrea Carolina Falconí Páez", "Andrés Sebastián Gudiño Vega", "María José Jaramillo Cartwright", "Sebastián Xavier Oña Vargas", "Sebastián Eduardo Puga Martínez" ], "abstract": "Background: Autism spectrum disorder (ASD) is a neurodevelopmental condition associated with an unclear etiologic mechanism. Following suggestions in the literature of a close relation between the gut microbiota and the central nervous system development, neuroimmune and neuroendocrine systems, new theories and strategies of the management of ASD in children focus on the brain-gut axis via microbiota transfer therapy. Despite the regular appearance in the news, the level of evidence supporting this intervention is unclear and to this date, no systematic review on this issue has been published. Methods: We conducted a systematic literature review of the efficacy and safety of microbiota transfer therapy for the management of ASD in children. MEDLINE via PubMed, LILACS IBECS via BVS, EMBASE via Ovid, Scopus and Cochrane Library were searched on 19th April 2018. Results: One single study published in 2017 was identified. The intervention group included 18 patients and showed significant clinical improvements in the gastrointestinal and ASD-related symptoms. The clinical procedure was reported as safe and well-tolerated with some transitory adverse effects. Conclusions: The causality and correlation of the intervention and the expected outcomes cannot be assumed with current evidence. In addition, recommendations about the effectiveness or safety of microbiota transfer therapy in children with ASD cannot be currently issued. Randomized controlled trials and clinical protocols for the intervention are needed.", "keywords": [ "microbiota transfer therapy", "autism spectrum disorder", "microbiome", "systematic literature review" ], "content": "Introduction\n\nAutism spectrum disorder (ASD) is a neurodevelopmental condition characterized by impaired communication, impaired reciprocal social interaction, and restricted, repetitive patterns of behavior or interests (Faras et al., 2010). In the past couple of decades, the prevalence of ASD has increased to around 1–2%, partially because of changes in the Diagnostic and Statistical Manual of Mental Disorders (DSM-V) diagnostic criteria and patients being diagnosed at a younger age (Park et al., 2016). Standardized research assessment tools have been designed following the DSM-IV criteria. Such tools include the Autism Diagnostic Interview-Revised (ADI-R) and the Autism Diagnostic Observation Schedule (ADOS) (Lyall et al., 2017).\n\nEtiologic theories of ASD have changed over the years. It is currently accepted that ASD is not a single-cause disorder, but rather a multi-factorial disorder associated with genetic and non-genetic risk factors involved. Causes related to genetics are estimated to be present in 10% to 20% of patients with ASD (Park et al., 2016). The specific biological mechanism remains unclear, although literature suggest a close relation between the gut microbiota and the central nervous system development, neuroimmune and neuroendocrine systems (Cani & Knauf, 2016)(Martin & Mayer, 2017), leading to the development of new theories and strategies for the management of ASD (Choi & Cho, 2016)(Yang et al., 2018). Children with ASD have been considered as a relevant interventional group for using microbiota transfer therapy. (Yang et al., 2018)(Cryan & Dinan, 2012).\n\nThe gut-brain axis describes a bidirectional communication between the gut microbiota and the central nervous system (Falsaperla et al., 2017). These bidirectional pathways control the permeability and barrier function of the gut, therefore, the composition of the microbiota. Probiotic agents, defined as living beings that positively influence health when ingested, and the gut microbiota can alter the concentration of cytokines and short-fatty acids (SCFA), which have a direct action in the brain function (Cryan & Dinan, 2012).\n\nIn the past decade, microbiota transfer therapy has gathered interest from research leading to the development of novel intervention for the management and clinical improvement of different pathologies, including ASD (Colman & Rubin, 2014). Microbiota transfer therapy aims to restore the gut microbiota of the receiver through the infusion of donor faeces to the gastrointestinal tract via oral capsules, endoscopic stomach, duodenum, jejunum, ileum, coecum or sigmoid infusions or rectal enema infusions (Rossen et al., 2015)(Choi & Cho, 2016). Recent evidence suggests that microbiota transfer therapy affects the pathophysiology of several brain disorders, such as ASD, Parkinson disease, chronic pain, and disorders in mood and affect (Martin & Mayer, 2017)(Yang et al., 2018). Microbiota transfer therapy has shown to cause the durable engraftment of donor microorganisms and increases the number of species present in the receiver’s gut microbiota (Kelly et al., 2015). There is already evidence showing its efficacy for different gastrointestinal diseases, such as inflammatory bowel disease, metabolic syndrome, irritable bowel syndrome, and Clostridium difficile infection (Rossen et al., 2015). As a novel therapy, there is still no protocol for using microbiota transfer therapy for the management of the different pathologies. However in 2010, a workgroup with several societies proposed a protocol for the treatment of Clostridium difficile infection with microbiota transfer therapy, including the indications, donor selection criteria, recipient exclusion criteria, means of administering stool, and the evaluation of the success (Bakken et al., 2011). There are still questions about the safety of the microbiota transfer therapy, as the possibility of transmitting infectious agents exists (Choi & Cho, 2016).\n\nTo this day, no systematic literature review of clinical trials, assessing the effects of microbiota transfer therapy in patients with ASD has been conducted. The aim of this study is to perform a systematic literature review on the efficacy and safety of the microbiota transfer therapy for the management of symptoms in children with ASD.\n\n\nMethods\n\nThis study was conducted following the 27 items of the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement to report a systematic review. No previous review protocol was identified for microbiota transfer therapy for the management of autism spectrum disorder in children. A protocol to identify and assess study eligibility was developed and was uploaded to the Dataverse repository as Appendix 1, Extended data (Estrella & Guillemot, 2019).\n\nSearch terms (see Appendix 1, Extended data (Estrella & Guillemot, 2019)) were developed based on other systematic literature reviews and adapted for each database (Rossen et al., 2015)(Colman & Rubin, 2014). References used in articles of peripheral relevance were reviewed. Searches were performed in five different databases: MEDLINE via PubMed, LILACS IBECS via BVS, EMBASE via Ovid, Scopus and Cochrane Library. The search was conducted on the 19th of April 2018.\n\nTo be included, the papers had to meet a set of eligibility criteria: Studies must be randomized controlled trials, quasi-randomized control trials or control trials. Eligible population was with children diagnosed with autism spectrum disorder (age limitation according to each study). The intervention was microbiota transfer therapy. For the data extraction, a standard data extraction form was used, which included authors, years, journal, number of patients included, eligibility criteria, study outcomes (efficacy and safety) and a final critical assessment of the trial. No language or publication date were used as exclusion criteria.\n\nEndNote version X7.7.1 was used to extract the information of captured citations and deduplication. Two reviewers independently assessed the titles and abstracts. Discrepancies were solved with discussion until a consensus was reached between reviewers. If the information of the database was not enough to use the selection criteria, the full manuscript was retrieved and reviewed in full.\n\nTo assess the risk of bias in estimates of the comparative effectiveness of the study in the included in this systematic literature review, the Risk Of Bias In Non-randomized Studies – of Interventions (ROBINS-I) was used (NCBI, n.d.; Sterne et al., 2016).\n\n\nResults\n\nIn total, 5297 papers were identified after automatic deduplication (see PRISMA flowchart, Figure 1). From those papers, one single study fulfilled all inclusion criteria.\n\nIdentification, screening, eligibility and inclusion of studies.\n\nThe study identified is an open-label trial investigating the safety, tolerability and efficacy of microbiota transfer therapy for gastrointestinal and behaviour symptoms in children with ASD and was published in 2017 (Kang et al., 2017). A total of 18 children participated, with ages ranging from seven to 16 years and that presented moderate to severe gastrointestinal problems, with 20 neurotypical children used as controls. After the microbiota transfer therapy intervention, children with ASD showed a greater bacteria diversity in the gut. The study showed substantial changes in gastrointestinal symptoms, such as constipation, diarrhoea, abdominal pain and indigestion. ASD symptoms showed significant improvements during the treatment and no reversion of the effects during the follow up with a decline of ASD symptoms of more than 20%. The average developmental age was found to be increased in 1.4 years with a p<0.001 with the VABS-II (Vineland Adaptive Behaviour Scale II) score. The study reported that microbiota transfer therapy was a safe and a well-tolerated intervention, with some transitory adverse effects, including hyperactivity and aggression during the first phase of vancomycin treatment. The overall bias associated with the study was considered low. While the risk of bias due to confounding domains was considered serious, the risk of bias in participant selection was considered low. The bias in measurement of outcomes was considered moderate.\n\n\nDiscussion\n\nTo date, this is the only published systematic literature review on microbiota transfer therapy on children with ASD. Although the intervention has been described as effective and safe in the study found, the 18 children of this study are not enough to make recommendations for future application of the microbiota transfer therapy intervention in ASD children. The causality and correlation of the intervention and the expected outcomes cannot be assumed with the current evidence.\n\nMost information available is not associated with randomized clinical trials. In a published observation, two children showed improvement in their ASD symptoms after faecal microbiota therapy and five children after several weeks of receiving cultured Bacteroidetes and Clostridia every day (Aroniadis & Brandt, 2013). In June 2019, the Food and Drug Administration (FDA) published a safety warning regarding the risk of adverse reactions after a microbiota transfer therapy that was not screened against an extended-spectrum β-lactamase-producing Escherichia coli (NEJM Journal Watch, 2019). The FDA added additional protections for investigational use of microbiota transfer therapy, that includes stool testing for multi-drug resistance organisms (MRDO) (Commissioner, 2019).\n\nThe brain-gut axis involves complex communication and metabolic mechanisms that are still not fully understood. Several pathophysiologic pathways have been proposed, hypothesizing the relation between the microbiota composition with different neurological conditions, nevertheless further evidence to clarify the function of this axis is needed. Taking in consideration that children with ASD present a pattern of dysbiosis, the microbiota transfer therapy has raised attention as a novel intervention for the management of ASD patients. However, it is still not clear if there is a direct relation between ASD and the alteration in their gut microbiota and therefore, if the change in the microbiota directly influences the different gastrointestinal and clinical symptoms of ASD. Besides, microbiota transfer therapy is a growing field in medicine for management of several diseases. It is not a new therapeutic modality however it has received public attention from the media in the past years. The first report of faecal material given to patients with gastroenterological illness was in China by Ge Hong (Aroniadis & Brandt, 2013). The use of faecal enemas was reported in 1958 as an adjunct treatment of pseudomembranous enterocolitis (Eiseman et al., 1958). Today, microbiota transfer therapy is extensively used for C. difficile infection, inflammatory bowel disease, and irritable bowel syndrome management (Sha et al., 2014). The evidence is growing around faecal microbiota therapy and its applications in other fields of medicine, including ASD patients. Efficacy and safety data have been recorded mainly in adults, while it has not been studied so widely in children.\n\nThe selection of microbiota transfer therapy donor’s is an important step to assure the safety of this intervention. There is not a standardized procedure for selection and screening of donors, and varies from study to study (Owens et al., 2013). The evaluation can include but is not limited to past medical history, questionnaires and a physical examination and serologic for bacteria, parasites and viral infections. Some studies have used exclusion criteria for donors, such as history of gastrointestinal pathologies, use of immunosuppressive drugs, prior use of antibiotics, and gastrointestinal symptoms (Paramsothy et al., 2015).\n\nThis systematic literature review shows that the evidence is not strong enough to make recommendations about the use of microbiota transfer therapy because of its effectiveness or safety in children with ASD. Randomized, placebo-controlled, double-blind control trials and clinical protocols for the intervention are needed. However new evidence is expected to be released as three clinical trials related to microbiota transfer therapy with ASD patients. Are currently online. One targets ASD and gastrointestinal disorders in children (The National Library of Medicine, 2019a), another focuses on adults (The National Library of Medicine, 2019b) and the third looks into children and young adults (The National Library of Medicine, 2019c).\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nHarvard Dataverse: Replication Data for: Efficacy and safety of microbiota transfer therapy for the management of Autism Spectrum Disorder in children: a systematic review. https://doi.org/10.7910/DVN/IFHLQB (Estrella & Guillemot, 2019).\n\nThis project contains the following underlying data:\n\nScreening Database (all studies initially identified in the search)\n\nAppendix 1 (Protocol and Search Terms for each Database)\n\nPRISMA Checklist (reporting guidelines)\n\nHarvard Dataverse: PRISMA checklist for ‘Efficacy and safety of microbiota transfer therapy for the management of autism spectrum disorder in children: a systematic review’. https://doi.org/10.7910/DVN/IFHLQB (Estrella & Guillemot, 2019).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors want to thank the review of Paul Cardenas, MD, PhD, professor of microbiology at Universidad San Francisco de Quito USFQ and the support of Michelle Grunauer, MD, PhD, Dean of Medicine, Universidad San Francisco de Quito USFQ.\n\n\nReferences\n\nAroniadis OC, Brandt LJ: Fecal microbiota transplantation: past, present and future. Curr Opin Gastroenterol. 2013; 29(1): 79–84. PubMed Abstract \| Publisher Full Text\n\nBakken JS, Borody T, Brandt LJ, et al.: Treating Clostridium difficile infection with fecal microbiota transplantation. Clin Gastroenterol Hepatol. 2011; 9(12): 1044–1049. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCani PD, Knauf C: How gut microbes talk to organs: The role of endocrine and nervous routes. Mol Metab. 2016; 5(9): 743–752. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChoi HH, Cho YS: Fecal Microbiota Transplantation: Current Applications, Effectiveness, and Future Perspectives. Clin Endosc. 2016; 49(3): 257–265. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nColman RJ, Rubin DT: Fecal microbiota transplantation as therapy for inflammatory bowel disease: a systematic review and meta-analysis. J Crohns Colitis. 2014; 8(12): 1569–1581. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCommissioner: Fecal Microbiota for Transplantation: Safety Communication- Risk of Serious Adverse Reactions Due to Transmission of Multi-Drug Resistant Organisms. FDA. 2019. Reference Source\n\nCryan JF, Dinan TG: Mind-altering microorganisms: the impact of the gut microbiota on brain and behaviour. Nat Rev Neurosci. 2012; 13(10): 701–712. PubMed Abstract \| Publisher Full Text\n\nEiseman B, Silen W, Bascom GS, et al.: Fecal enema as an adjunct in the treatment of pseudomembranous enterocolitis. Surgery. 1958; 44(5): 854–859. PubMed Abstract\n\nEstrella P, Guillemot J: Replication Data for: Brief report: Efficacy and safety of microbiota transfer therapy for the management of Autism Spectrum Disorder in children: a systematic review [Data set]. 2019. http://www.doi.org/10.7910/DVN/IFHLQB\n\nFalsaperla R, Romano C, Pavone P, et al.: The Gut-brain Axis: A New Pathogenic View of Neurologic Symptoms - Description of a Pediatric Case. J Pediatr Neurosci. 2017; 12(1): 105–108. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFaras H, Al Ateeqi N, Tidmarsh L: Autism spectrum disorders. Ann Saudi Med. 2010; 30(4): 295–300. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKang DW, Adams JB, Gregory AC, et al.: Microbiota Transfer Therapy alters gut ecosystem and improves gastrointestinal and autism symptoms: an open-label study. Microbiome. 2017; 5(1): 10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKelly CR, Kahn S, Kashyap P, et al.: Update on Fecal Microbiota Transplantation 2015: Indications, Methodologies, Mechanisms, and Outlook. Gastroenterology. 2015; 149(1): 223–237. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLyall K, Croen L, Daniels J, et al.: The Changing Epidemiology of Autism Spectrum Disorders. Annu Rev Public Health. 2017; 38: 81–102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMartin CR, Mayer EA: Gut-Brain Axis and Behavior. In E. Isolauri, P. M. Sherman, & W. A. Walker (Eds.), Nestle Nutr Inst Workshop Ser. S. Karger AG. 2017; 88: 45–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNCBI: Appendix F. Cochrane Risk of Bias Tool. National Center for Biotechnology Information. Cochrane Risk of Bias Tool - NCBI, (n.d.). Reference Source\n\nNEJM Journal Watch: Patient Dies After Receiving Fecal Transplant That Contained Drug-Resistant Bacteria. NEJM Journal Watch. 2019; 2019. Reference Source\n\nOwens C, Broussard E, Surawicz C: Fecal microbiota transplantation and donor standardization. Trends Microbiol. 2013; 21(9): 443–445. PubMed Abstract \| Publisher Full Text\n\nParamsothy S, Borody TJ, Lin E, et al.: Donor Recruitment for Fecal Microbiota Transplantation. Inflamm Bowel Dis. 2015; 21(7): 1600–1606. PubMed Abstract \| Publisher Full Text\n\nPark HR, Lee JM, Moon HE, et al.: A Short Review on the Current Understanding of Autism Spectrum Disorders. Exp Neurobiol. 2016; 25(1): 1–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRossen NG, MacDonald JK, de Vries EM, et al.: Fecal microbiota transplantation as novel therapy in gastroenterology: A systematic review. World J Gastroenterol. 2015; 21(17): 5359–71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSha S, Liang J, Chen M, et al.: Systematic review: faecal microbiota transplantation therapy for digestive and nondigestive disorders in adults and children. Aliment Pharmacol Ther. 2014; 39(10): 1003–1032. PubMed Abstract \| Publisher Full Text\n\nSterne JA, Hernán MA, Reeves BC, et al.: ROBINS-I: a tool for assessing risk of bias in non-randomised studies of interventions. BMJ. 2016; 355: i4919. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe National Library of Medicine: Efficacy, Safety, and Tolerability Study of Oral Full-Spectrum MicrobiotaTM (CP101) in Subjects With Autism Spectrum Disorder and Associated GI Symptoms (SPROUT)—Full Text View—ClinicalTrials.gov. 2019a. Reference Source\n\nThe National Library of Medicine: Microbiota Transfer Therapy for Adults With Autism Spectrum Disorder (ASD) Who Have Gastrointestinal Disorders—Full Text View—ClinicalTrials.gov. 2019b. Reference Source\n\nThe National Library of Medicine: The Gut-Brain Study—Full Text View—ClinicalTrials.gov. 2019c. Reference Source\n\nYang Y, Tian J, Yang B: Targeting gut microbiome: A novel and potential therapy for autism. Life Sci. 2018; 194: 111–119. PubMed Abstract \| Publisher Full Text" }	[ { "id": "59156", "date": "13 Feb 2020", "name": "Valerie d'Astous", "expertise": [ "Reviewer Expertise Autism" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important and timely systematic review of the efficacy and safety of microbiota transfer therapy (MTT) use for autism spectrum disorder in children. MTT is a current subject of interest and controversy among researchers and clinicians. Thorough and unbiased, this systematic review’s findings provide a very limited evidence base of one study; with early outcomes and a small sample size warranting cautious interpretation of the findings. Moreover, the authors note that MTT is still considered an experimental treatment with the FDA warning of unknown risk factors and specifying safety guidelines for donor recruitment and assessments of treatments.\n\nThis paper would have benefited from a more detailed discussion and critique of the one study identified. In particular, clarification of the low bias stated in the study’s results required greater discussion. For example, an explanation of the ‘open label’ methodology, which does not include a blinded comparison group and has been demonstrated to result in a potentially high placebo effect seems to contradict the low risk of bias reported. A greater critique of the study would have strengthened and supported the authors concluding statements for the need for randomized, placebo-controlled, double-blinded control trial. Additionally, while the authors accurately state that causality and correlation of the intervention and outcomes cannot be assumed based on current evidence, a more detailed discussion of confounding factors in this study may have raised further evidence-based limitations.\n\nThis procedurally detailed, well written systematic review appropriately concludes that more specific and controlled research is needed to determine the safety and efficacy of MTT as an intervention modality for autistic children with moderate to severe gastrointestinal problems. Furthermore, it provides an opportune contribution to this subject matter.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] }, { "id": "59159", "date": "10 Mar 2020", "name": "Gianluca Ianiro", "expertise": [ "Reviewer Expertise microbiota" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a systematic review of the efficacy and safety of microbiota transfer therapy for the management of ASD in children.\n\nThe following databases have been searched up to April 2018: MEDLINE via PubMed, LILACS IBECS via BVS, EMBASE via Ovid, Scopus and Cochrane Library.\n\nThe authors found only one study published in 2017 (18 patients), that fitted the inclusion criteria. With one study, it is not possible to index this paper.\n\nI would suggest to re-do the search, updating it to March 2020 (last search was in April 2018). If other papers (at least 2 beyond the one already identified) will be found, so it could be worth to index.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-48
https://f1000research.com/articles/9-45/v1	24 Jan 20	{ "type": "Research Article", "title": "Video-augmented vs standard consent in an early ICU cycling feasibility trial: a randomized embedded recruitment trial", "authors": [ "Michelle E. Kho", "Mark Duffett", "France J. Clarke", "Melissa Shears", "Alexander J. Molloy", "Deborah J. Cook", "Mark Duffett", "France J. Clarke", "Melissa Shears", "Alexander J. Molloy", "Deborah J. Cook" ], "abstract": "Background: In a trial of early in-bed cycling in critically ill patients, a video demonstrating use of the cycle in addition to verbal description may improve satisfaction with the informed consent process for all persons involved. Methods: A convenience sample of in-person consent encounters for enrolment in TryCYCLE (NCT01885442), a 33-patient pilot study of in-bed cycling with mechanically ventilated patients in an intensive care unit, were recruited. In this study within a trial, using concealed allocation, we randomized consent encounters to a Video or Standard consent approach. Those in the Video group viewed a 2-minute video of a model using in-bed cycling plus the routine verbal description of the study. The Standard group received the routine verbal description only. Patients and/or substitute decision makers (SDMs) were blinded to the study purpose. After each encounter, patients and/or SDMs and the research coordinator submitted written satisfaction and comfort ratings using 7-point scales (higher scores better). We documented consent outcomes and analyzed between group differences with independent group t-tests. Results: We randomized 14 encounters (6 Video, 8 Standard). Ten completed questionnaires (5 in each group) demonstrated no difference in patient and/or SDM satisfaction or comfort between Video or Standard (mean [standard deviation] Satisfaction: 6.8[0.45] vs. 7.0[0] vs. p=0.37; Comfort: 7.0[0] vs. 7.0[0], p>0.99). The research coordinator evaluated all randomized encounters, with no differences between Video or Standard (Satisfaction: 7.0[0] vs. 6.9[0.35], p=0.41; Comfort: 6.7[0.52] vs. 6.9[0.35], p=0.39). All 14 consent encounters enrolled in TryCYCLE. Conclusions: Patient and/or SDM satisfaction and comfort with consent was very high for both the Video and Standard approaches. Further research, including use of videos to portray different study interventions, is needed, including analysis of patient and/or SDM satisfaction, comfort, comprehension, and consent rates. Registration for host trial: ClinicalTrials.gov, NCT01885442, registered on June 25, 2013", "keywords": [ "Evidence-based Medicine", "Randomized controlled Trials", "Research design", "Informed consent", "Clinical trials", "Audiovisual aids", "mechanical ventilation", "cycle ergometry", "Studies within a trial" ], "content": "Introduction\n\nIn-bed cycling is a novel critical care rehabilitation intervention that could improve patients’ physical function1. In-bed cycling is not commonly used in the intensive care unit (ICU)2 and is likely unfamiliar to many patients and their substitute decision makers (SDMs). A video demonstrating the use of the cycle in addition to verbal informed consent may improve their satisfaction and comfort with the consent process. The purpose of this study within a trial (SWAT) was to determine the effect of video-augmented versus standard informed consent on patient and SDM satisfaction and comfort.\n\n\nMethods\n\nThe study protocol is available as Extended data3.\n\nWe included a convenience sample of consent encounters in TryCYCLE (NCT01885442), a 33-patient, single-centered, safety and feasibility study of in-bed cycling in critically ill patients4. The TryCYCLE study required a priori informed consent. Patients and SDMs who were approached for in-person informed consent for TryCYCLE in our mixed medical-surgical ICU (St Joseph’s Healthcare ICU) in Hamilton, ON were eligible for this substudy. We excluded encounters where initial consent was received by phone, as respondents could not view the video as part of their initial deliberations.\n\nUsing concealed allocation with variable undisclosed block size, we randomized consent encounters to receive augmented video consent in addition to verbal consent discussion (Video) or verbal consent discussion only (Standard) in a 1:1 ratio. To maintain allocation concealment, we used third-party assignment. An individual not otherwise involved in the study (MD) prepared a computer-generated randomization sequence and provided the group assignment immediately prior to each consent encounter.\n\nThe research coordinator used a tablet computer to show and describe how an ICU patient would bike in the TryCYCLE study. The 2-minute video included footage of a model on the bike and an ICU patient biking while mechanically ventilated. The research coordinator also provided a verbal description of the research study, which was not scripted to allow for spontaneous exchange. The research coordinator and patient and/or SDM had the consent encounter guided by the usual principles of informed consent for ICU studies previously published5. The video did not include sound.\n\nComparison: Verbal description only.\n\nOur primary outcomes were patient and/or SDM satisfaction and comfort with the consent encounter. Secondary outcomes included patient and/or SDM perception of video helpfulness, and research coordinator assessments of satisfaction with the consent process, their own comfort with the consent encounter, patient and/or SDM study comprehension, and the number and perceived difficulty of questions asked by patient and/or SDM. Respondents assessed outcomes after the consent encounter using 7-point Likert-type scales, with higher scores representing more favorable ratings (see Extended data3). We invited patients and/or SDMs to return the 1-page, self-administered questionnaire within 24 hours in a pre-addressed, sealable envelope. The research coordinator documented consent outcome and duration of the encounter.\n\nPatients and SDMs were not aware of the comparison study at the time of the consent encounter. Both groups received the same consent forms for the TryCYCLE study3. The analyst was blinded to treatment group.\n\nWe did not calculate a sample size a priori for this SWAT. We reported continuous variables as mean (standard deviation [SD]) and categorical values as count (percent). We used Student’s t-tests to compare the results of the two groups, Pearson’s Chi Square for categorical comparisons, and p=0.05 as the criterion for statistical significance. We conducted all analyses with SPSS version 25.0 (IBM, Armonk, New York).\n\nThis study, and the host study (TryCYCLE) were approved by the Hamilton Integrated Research Ethics Board (HIREB 13-173), which waived the need for consent for this substudy. Study participants could opt-out of this substudy by declining to return the follow-up questionnaire.\n\n\nResults\n\nWe randomized 14 of the 37 consent encounters for TryCYCLE between October 2013 and August 2014: 6 to Video and 8 to Standard (Figure 1). Table 1 summarizes the characteristics of the convenience sample in this study. We received 10 completed questionnaires (5 from each group), which contributed to estimates of effect. We report all comparisons as Video vs. Standard. Patient and/or SDM rating of satisfaction was not different between groups, the mean [SD] was 6.8 [0.5] vs. 7.0 [0] (p=0.37). Patient and/or SDM rating of comfort was the same for both groups (7.0 [0], p>0.99). Patient and/or SDM rating of video helpfulness was 6.6 [0.54]. All 14 (100%) patients and/or SDMs provided consent to participate in the TryCYCLE study (the overall consent rate for TryCYCLE was 91.9%)4.\n\nOf the 23 non-randomized patients who had consent encounters, 12 occurred by phone, 5 were in-person by intubated patients who were unable to complete the feedback form, 3 declined participation in the host study and these consent encounters occurred without the option for enrolment in the SWAT, 2 in-person consents were obtained by a consultant intensivist, and in 1 instance we already initiated the consent discussion before consideration of randomization in this trial. Post-randomization, 1 person was not offered the feedback form due to perceived emotional distress by the research coordinator.\n\nSDM= substitute decision maker.\n\nOf the 14 consent encounters rated by the research coordinator, satisfaction (7.0 [0] vs. 6.9 [0.35], p=0.41) and comfort (6.7 [0.52] vs. 6.9 [0.35], p=0.39) were similar. There was no difference in perceived patient and/or SDM understanding of the research study (6.67 [0.51] vs. 7.0 [0] vs., p=0.11), number of questions asked (1-5 questions: 6 (75.0%) vs. 5 (83.3%), p=0.66), nor perceived difficulty of clarifying questions (2.67 [1.86] vs. 1.86 [1.4], p=0.40). The encounter duration was not different (18.3[14.7] vs. 13.3[3.7] minutes, p=0.20).\n\n\nDiscussion\n\nIn this small randomized study of video-augmented versus standard consent for in-bed cycling in the ICU, there was no difference in satisfaction or comfort between groups. Our results are similar to previous studies included in a systematic review of audiovisual plus standard versus standard informed consent, identifying no difference in consent rate between groups6. Given the universally high ratings for satisfaction and comfort by consent recipients, we hypothesize that video-augmented consent could be useful for describing more complex interventions or facilitating conversations when research centres or coordinators are new to conducting clinical trials. Involving patient-family advisory groups may help guide use and content of video adjuncts for future consent encounters.\n\nOur study also contributes to global efforts to study efficiencies in trial conduct through SWATs7. It directly addresses Priority 4 (What are the best approaches for designing and delivering information to members of the public who are invited to take part in a randomized trial) of the top 10 trial recruitment issues informed by stakeholders, including members of the public, front line clinical and research staff, trial principal investigators, and funders8.\n\nOur study has limitations, principally the small sample size and significant expertise in consent discussions from a single research coordinator with 20 years of experience in critical care studies. We did not measure consent recipient knowledge. Our study also has several strengths. Patients and/or SDMs were blinded to the Video vs Standard comparison study. We evaluated practical aspects of the consent process, focusing on outcomes we anticipate are important to patients and SDMs. We addressed gaps in previous research by measuring SDM and clinical researcher satisfaction, and time to administer the video-augmented consent6.\n\n\nConclusions\n\nOverall, patient, SDM, and research coordinator satisfaction and comfort with consent was very high for both the video-augmented and standard approaches. Further research, including use of videos to portray different study interventions, is needed, including analysis of patient and/or SDM satisfaction, comfort, comprehension, and consent rates.\n\n\nData availability\n\nSince the intervention video includes an identifiable critically ill patient, and permission to publish this was not obtained from the patient, patient’s family or the research ethics board, this cannot be shared.\n\nOpen Science Framework: Video-augmented vs. standard consent in an early ICU cycling feasibility trial: a randomized embedded recruitment trial, https://doi.org/10.17605/OSF.IO/4SPGA3\n\nThis project contains the following underlying data:\n\nTryCYCLE Video RCT data-SDM CRF.csv: data corresponding to data collection forms for patient/SDM\n\nTryCYCLE Video RCT data-RC CRF.csv: data corresponding to data collection forms for research coordinator\n\nOpen Science Framework: Video-augmented vs. standard consent in an early ICU cycling feasibility trial: a randomized embedded recruitment trial, https://doi.org/10.17605/OSF.IO/4SPGA3\n\nThis project contains the following extended data:\n\nTryCYCLE Video consent study protocol.docx\n\nTryCYCLE Video consent data collection forms.docx\n\nOpen Science Framework: CONSORT checklist for ‘Video-Augmented vs Standard Consent in an Early ICU Cycling Feasibility Trial: A Randomized Embedded Recruitment Trial’, https://doi.org/10.17605/OSF.IO/4SPGA3\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nBurtin C, Clerckx B, Robbeets C, et al.: Early exercise in critically ill patients enhances short-term functional recovery. Crit Care Med. 2009; 37(9): 2499–505. PubMed Abstract \| Publisher Full Text\n\nKoo KK, Choong K, Cook DJ, et al.: Early mobilization of critically ill adults: a survey of knowledge, perceptions and practices of Canadian physicians and physiotherapists. CMAJ Open. 2016; 4(3): E448–E54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKho M: Video-augmented vs. standard consent in an early ICU cycling feasibility trial: a randomized embedded recruitment trial. 2020. http://www.doi.org/10.17605/OSF.IO/4SPGA\n\nKho ME, Molloy AJ, Clarke FJ, et al.: TryCYCLE: A Prospective Study of the Safety and Feasibility of Early In-Bed Cycling in Mechanically Ventilated Patients. PLoS One. 2016; 11(12): e0167561. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith OM, McDonald E, Zytaruk N, et al.: Enhancing the informed consent process for critical care research: strategies from a thromboprophylaxis trial. Intensive Crit Care Nurs. 2013; 29(6): 300–309. PubMed Abstract \| Publisher Full Text\n\nSynnot A, Ryan R, Prictor M, et al.: Audio-visual presentation of information for informed consent for participation in clinical trials. Cochrane Database Syst Rev. 2014; (5): CD003717. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTreweek S, Bevan S, Bower P, et al.: Trial Forge Guidance 1: what is a Study Within A Trial (SWAT)? Trials. 2018; 19(1): 139. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHealy P, Galvin S, Williamson PR, et al.: Identifying trial recruitment uncertainties using a James Lind Alliance Priority Setting Partnership - the PRioRiTy (Prioritising Recruitment in Randomised Trials) study. Trials. 2018; 19(1): 147. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "59119", "date": "28 Jan 2020", "name": "David J. Torgerson", "expertise": [ "Reviewer Expertise Interest in SWATS and their conduct as well as being the Director of Trials Unit" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting SWAT, which despite its small size is of interest. A few suggestions to improve the reporting:\n\nHave you registered the SWAT with the Northern Ireland Hub for Trials Methodology Research (https://www.qub.ac.uk/sites/TheNorthernIrelandNetworkforTrialsMethodologyResearch/SWATSWARInformation/Repositories/SWATStore/) this will allow others to replicate the study as this is obviously required.\n\nThe reporting of the means should also include the differences and 95% confidence intervals of the differences.\n\nYou used block randomisation - what block sizes were used?\n\nWith a tiny sample size, I think some qualitative outcomes, if collected, would enhance the findings of the study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "63783", "date": "01 Jun 2020", "name": "Thierry Boulain", "expertise": [ "Reviewer Expertise Intensive care medicine", "clinical research" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study within a trial, the authors included surrogate decision-makers (SDM) approached for potential consent to the enrollment of patients in a randomized trial. The aim of this trial within a trial was to compare the satisfaction and comfort of the SDM (and of patients, but finally very few patients were included) regarding the consent encounter, between two groups who were randomly allocated to video-augmented consent encounter or standard consent encounter. The main findings are that SMD reported similar satisfaction and comfort scores in both groups. Finally, only 5 subjects in each group could be studied.\n\nThe obvious, main weakness of this study is its very small sample size. This has several consequences: First, estimates of outcome measures (satisfaction and comfort scores) are imprecise; Second, comparison tests between groups have very low power; Third, the authors conclude that comfort score was very high in both groups. Indeed all SDM gave a score of 7 on the 7-point Likert scale. Imagine the “very high score” was defined as a score of 7. In the convenience sample studied, the proportion of patients with very high score was 100% (5/5) in each group. This only tells us that the proportion of high score in the population is between 57% and 100% (95% Wilson confidence interval) (and perhaps close to 57% in one group and close to 100% in the other group). Is a very high score proportion of 57% satisfactory? This is a qualitative judgment, but I would say no.\n\nScores on Likert scales are presented as means and SD and compared using parametric test. This assumes that the Likert scale is a homogeneous continuous scale where the distance or the increase in comfort is the same between each point, and that the distance between, say point 4 and point 6, is two times the distance between point 1 and point 2. This assumption is likely not true. For this reason, I would give median and percentiles to report scores and perform between-group comparison using a nonparametric test (Mann-Withney U test). Also, given the small samples studied, presenting all individual data, in boxplots, for example, would give an honest picture of what was observed.\n\nAs explained above, and although the authors were cautious in referring only to “high scores” in their conclusion, even this conclusion is not fully supported by the observed data.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-45
https://f1000research.com/articles/8-23/v1	07 Jan 19	{ "type": "Research Article", "title": "Factors associated with self-medication in users of drugstores and pharmacies in Peru: an analysis of the National Survey on User Satisfaction of Health Services, ENSUSALUD 2015", "authors": [ "Diego Urrunaga-Pastor", "Vicente A. Benites-Zapata", "Edward Mezones-Holguín", "Diego Urrunaga-Pastor", "Edward Mezones-Holguín" ], "abstract": "Background: Irresponsible self-medication is a problem for health systems in developing countries. We aimed to estimate the frequency of self-medication and associated factors in users of drugstores and pharmacies in Peru. Methods: We performed a secondary data analysis of the 2015 National Survey on User Satisfaction of Health Services (ENSUSALUD), a two-stage probabilistic sample of all regions of Peru. Non self-medication (NSM), responsible self-medication (RSM) and irresponsible self-medication (ISM) were defined as the outcome categories. Demographic, social, cultural and health system variables were included as covariates. We calculated relative prevalence ratios (RPR) with their 95% confidence intervals (95%CI) using crude and adjusted multinomial logistic regression models for complex samples with NSM as the referent category. Results: 2582 participants were included. The average age was 41.4 years and the frequencies of NSM, RSM and ISM were 25.2%, 23.8% and 51.0%; respectively. The factors associated with RSM were male gender (RPR: 1.35; 95%CI: 1.06-1.72), being between 40 and 59 years old (RPR: 0.53; 95%IC: 0.39-0.72), being 60 or older (RPR: 0.39; 95%IC: 0.25-0.59), not having health insurance (RPR: 1.89; 95%CI: 1.31-2.71) and living in the Highlands region (RPR: 2.27; 95%CI: 1.23-4.21). The factors associated with ISM were male gender (RPR: 1.41; 95%CI: 1.16-1.72), being between 40 and 59 years old (RPR: 0.68; 95%IC: 0.53-0.88), being 60 or older (RPR: 0.65; 95%IC: 0.48-0.88) and not having health insurance (RPR: 2.03; 95%CI: 1.46-2.83). Conclusion: Around half of the population practiced ISM, which was associated with demographic and health system factors. These outcomes are the preliminary evidence that could contribute to the development of health policies in Peru.", "keywords": [ "Adults", "Pharmacies", "Self-Medication", "Universal Coverage", "Insurance", "Health Services Accessibility", "Peru" ], "content": "Introduction\n\nSelf-medication is a practice that represents a public health problem worldwide1, mainly in developing countries, where it is an important issue for the health systems. The World Health Organization (WHO) defines self-medication as the selection and use of medicines by individuals to treat self-recognized illness or symptoms2. In addition, the concept of responsible self-medication (RSM) is based on the treatment of diseases and conditions using medicines that do not require a prescription for their sale, due to their safety and effectiveness when correctly used2. For that reason, there are over the counter (OTC) medicines, which would respond to the concept of RSM3. To study these behaviors is relevant in developing countries of Latin America.\n\nThe frequency of self-medication differs according to the country and context evaluated. Studies have reported self-medication prevalence ranging from 27% to 90.1%. In Asia, a study made in India reported a prevalence of 71%4, while in Iran it was 35.4%5. In Europe, research studies in Spain reported a prevalence between 14% and 90.1%6–8. In Latin America, Colombian studies presented prevalence ranges from 27.3% to 55.4%9–11, whereas in Brazil, it oscillated from 31% to 86.4%12,13. In Peru, a previous work found a self-medication prevalence of 56.7% in an urban area of Lima14. Then, we consider relevant to research the prevalence of self-medication since it has benefits and risks15.\n\nSelf-medication practice can entail suffering serious adverse effects. In addition, the concomitant use of several medicines may develop interactions that could increase those adverse effects1,16. Even OTC medicines used inappropriately and irresponsibly can represent a risk for the consumer1,15,17. Among the main benefits of the RSM, we can mention the increased access to pharmaceutical products, the reduction of unnecessary medical appointments, and of the expenses in healthcare services by the government15. For this reason it is important to evaluate the factors associated with the irresponsible self-medication practice (ISM)18.\n\nAmong the main conditions associated with self-medication practice are demographic, social, cultural, personal and health system factors. Age, sex, socio-economic status and educational level are frequently related to self-medication practice10. Among the personal factors associated with self-medication are having good results after self-medication, the belief of having experienced manageable similar symptoms previously, the fear of being diagnosed with a serious disease and the need to alleviate symptoms prior using healthcare services16,19,20. Regarding healthcare system, it has been described that self-medication is related to the easy access to medicines in drugstores and pharmacies and the lack of access to healthcare services, which in turn, is associated with the lack of a health insurance19. Thus, it is proved the multifactorial character of the self-medication practice.\n\nIn this context, despite its relevance, we have not found nationwide studies that had evaluate the frequency of self-medication in users of drugstores and pharmacies in Peru. The objective of this study was to estimate the frequency of RSM and ISM, and to identify the factors associated with such practice in a population-based sample.\n\n\nMethods\n\nWe performed a secondary data analysis using the fourth questionnaire of the National Survey on User Satisfaction of Health Services (ENSUSALUD) of 2015. ENSUSALUD is an annual questionnaire, the first edition was carried out in 2014, and was applied to internal and external users of healthcare facilities. The survey has been executed by the National Institute of Statistics and Informatics (INEI, by its Spanish initials) in collaboration with the National Superintendency of Health (SUSALUD, by its Spanish initials)21.\n\nENSUSALUD is composed by six questionnaires, the fourth questionnaire evaluated the drugstores and pharmacies users who were within the perimeter of two blocks around the 181 healthcare facilities that provided health services for the Ministry of Health and Regional Government (MINSA-GR, by its Spanish initials), Social Security System (EsSalud, by its Spanish initials), Health Service of the Armed Forces and Police (FF.AA.PP, by its Spanish initials) and Private Practice (CSP, by its Spanish initials) evaluated in the first and second questionnaires21. The study was carried out in 179 drugstores and pharmacies in the 25 regions of Peru.\n\nThe population was composed of clients of drugstores and pharmacies, who were surveyed after the purchase of a medicine. In total, the fourth questionnaire included 3863 participants. The sample size calculated of 3,863 participants represented an expanded population of 3,078,419 people; participants were not excluded due to lack of data or incomplete records (Figure 1). The calculation of the sample size used a design effect of 1.2 and a satisfaction possibility of 30% according to the results of the survey ENSUSALUD 2014; assuming a confidence level of 95%. Since our study was a secondary analysis, we calculate the statistical power and the result was 99%.\n\nThe sampling was probabilistic, stratified, for each one of the 25 regions of Peru, and two-staged. Drugstores and pharmacies were considered as primary sampling units, while users who went to such establishments were the secondary sampling units.\n\nThe fourth questionnaire of ENSUSALUD included people who went and bought a medicine for themselves, their kid(s) or partner in a pharmacy or drugstore close to a health care establishment. For this study, we only included people who have bought medicine for themselves, and this was specified in the question (c4pa): Who did you buy medicine(s) for? Marking the alternative: “for myself”, so the self-medication definition of the WHO was met2.\n\nWe excluded from the analysis the adults that bought medicines for their kid(s) or partner, who has not been able to go to the pharmacy and drugstore, since it was not possible to obtain the sociodemographic data and variables of interest of those people. A total of 1,226 (31.7%) people were excluded since they did not buy medicines for themselves (for their kid or partner). In addition, 55 participants were excluded for being under 18 years old. Finally, a total of 2,582 people were included in the study and they represented an expanded population of 2,057,592 people (Figure 1). Despite the exclusion of 1,281 (33.2%) participants, the number of participants in the strata of the variables studied did not generated statistical differences.\n\nResponse variable. We used the question (c4p12): “These medicines, did you buy them with prescription?” to define the response variable. The answers were divided into three options: yes, and showed prescription=1; yes, and did not show prescription =2; no=3. Based on these categories, we created a dichotomous variable called self-medication (yes=1 and no=0), the self-medication category included those participants who bought medicines without doctor’s prescription and those who did not show the prescription when they were surveyed. Additionally, we categorized those participants who had self-medicated in two strata according to the type of medicine they bought (c4p11_1e): RSM (with OTC medicines) and ISM (without using OTC medicines). On the other hand, the non self-medication (NSM) category was composed of participants who bought medicines and showed doctor’s prescription when they were surveyed. Consequently, three final categories were generated (NSM=0; RSM=1; ISM=2).\n\nExposure variables.\n\nDemographic, social and cultural factors\n\nWe included the following variables: sex (c4p3), age (c4p1), language (c4p5), education level (c4p4), current occupation (c4p6), guidance or help for self-medication (c4p21) and geographic region of residency (dominiog).\n\nFactors associated with the Health System\n\nWe included the following variables: health insurance affiliation (c4p7), type of health insurance (c4p8) and the request of prescription by the pharmacist when buying the medicine (c4p19).\n\nENSUSALUD 2015 is publicly accessible: http://portal.susalud.gob.pe/blog/base-de-datos-2015/. We downloaded the database without identifiers; thus, the confidentiality of the information given by the participants was guaranteed. Data collection was carried out after the verbal consent of the participants, it did not involve the biological sampling and was conducted for the management of health services nationwide.\n\nThe database was downloaded from SUSALUD’s website in compatible format with the statistical package STATA ® v14.0 (Stata Corporation, College Station, Texas, USA). The database was programmed for a complex sample analysis, the regions of Peru were considered as strata; and drugstores, pharmacies and their users were considered as sampling units. The STATA module “complex survey data” (svy) was used.\n\nThe categorical variables were shown as absolute frequencies and as weighted proportions by the complex sampling, with their respective 95% confidence intervals (95%CI). Weighted proportions were calculated which allowed a comparison between the variable’s categories included in the analysis. In this way, we evaluated the association between variables, RSM and ISM practice through Pearson’s chi-squared test corrected for design purposes.\n\nTo evaluate the factors associated with RSM and ISM, multinomial logistic regression models were conducted (crude and adjusted) and the complex sampling of the study was considered (svy)22. NSM was considered as the reference category.\n\nThe variables that showed statistically significant association (p<0.05) in the bivariate analysis, were included in the multinomial regression model. Possible collinearity relationships among variables was evaluated to obtain an adequate statistical consistency in the adjusted model. We developed a variable based on health insurance affiliation in participants (c4p7) and the type of health insurance (c4p8). This variable was evaluated in the multinomial regression models (crude and adjusted). The measure of association reported was the relative prevalence ratio (RPR), with their respective 95%CI. Moreover, we elaborated a second multivariate model that included variables whose association with self-medication has been described in the literature. However, this was similar to the first model prepared.\n\n\nResults\n\nWe found that 57.4% participants were women, the average age was 41.4, 96.7% of the respondents spoke Spanish and only 25.3% of the participants had university education. Likewise, 69.4% of the respondents were affiliated to a health insurance, of which more than half were covered by the Comprehensive Health Insurance (SIS, by its Spanish initials) (52.8%) and EsSalud (40.0%).\n\nThe prevalence of NSM, RSM, and ISM was 25.2%, 23.8%, and 51.0% respectively. Only 27.7% of the participants were asked for their prescription by the pharmacist when buying the medicine. Furthermore, 54.6% of the participants received guidance or help by the drugstore or pharmacy personnel to self-medicate, and 13.1% of the participants resided in Lima (Table 1).\n\n* Weight proportions and design effect of complex survey sampling were included.\n\n§ Refers to complete or incomplete university, non-university higher education, or high school education.\n\n& Refers only to users who had health insurance.\n\nWhen analyzing the total number of self-medicated users, it was found that the most commonly purchased medicine were non-steroidal anti-inflammatory drugs (NSAIDs) (24.4%), followed by antibiotics (16.5%) and analgesics/antipyretics/corticoids (16.4%). Also, the types of drugs most commonly purchased by participants who irresponsibly self-medicated were: NSAIDs (24.0%), antibiotics (22.6%) and gastrointestinal drugs (15.3%) (Figure 2).\n\nAfter evaluating the 651 participants who did not self-medicate, it was found that the main drugs purchased by this group were antibiotics (26.1%), NSAIDs (16.7%) and gastrointestinal drugs (9.5%) (Figure 2).\n\nThe absolute number of users per category of the variables studied was shown, in addition to the RSM and ISM percentages per each category. The corresponding weighting was considered.\n\nNo significant association was found between the practice of self-medication and language. However, there was statistically significant association with sex, age, education level, current occupation, having a health insurance, health insurance type, the request for prescription by the pharmacist when buying the medicine, guidance or help for self-medication, and region of residence (Table 2).\n\n* Weight proportions and design effect of complex survey sampling were included.\n\n** It included the following categories: Health Insurance from Private Companies, Health Insurance from Private Clinics, College student health insurance, FF.AA.PP. Insurance.\n\n§ Refers to complete or incomplete university, non-university higher education, or high school education.\n\n† It refers to the statistical significance obtained from the comparison of proportions between categories of the variable considering the complex survey sampling.\n\nIn the crude analysis, there was a greater RSM and ISM frequency in relation to male gender, current occupation (being a student) and not having a health insurance. Living in the Highlands region was associated with a higher frequency of RSM. Likewise, there was less frequency of RSM and ISM in relation to age (40 to 59 years and 60 to older), education level (complete elementary education or below) and current occupation (housewife) (Table 3).\n\n* A multinomial logistic regression model was performed considering the weighted proportions and design effect of the complex survey sampling.\n\n** It included the following categories: Health Insurance from Private Companies, Health Insurance from Private Clinics, College student health insurance, FF.AA.PP. Insurance.\n\n§ Refers to complete or incomplete university, non-university higher education, or high school education.\n\n§§ Not included in the adjusted model due to collinearity with gender and type of medical insurance.\n\nThe factors associated with RSM in the adjusted analysis were male gender (RPR: 1.35; 95%CI: 1.06-1.72), not having health insurance (RPR: 1.89; 95%CI: 1.31-2.71) and living in the Highlands region (RPR: 2.27; 95%CI: 1.23-4.21). On the other hand, the only factors that remained associated with a lower frequency of RSM were being between 40 and 59 years old (RPR: 0.53; 95%IC: 0.39-0.72) and being 60 or older (RPR: 0.39; 95%IC: 0.25-0.59) (Table 3).\n\nThe factors associated with a higher frequency of ISM in the adjusted analysis were male gender (RPR: 1.41; 95%CI: 1.16-1.72) and not having health insurance (RPR: 2.03; 95%CI: 1.46-2.83). Furthermore, the factors that remained associated with a lower frequency of ISM were being between 40 and 59 years old (RPR: 0.68; 95%IC: 0.53-0.88) and being 60 or older (RPR: 0.65; 95%IC: 0.48-0.88) (Table 3).\n\n\nDiscussion\n\nThis study found that three-quarters of the participants self-medicated and one out of two of the total population practiced ISM. Also, two out of three participants who irresponsibly self-medicated were not asked for the corresponding prescription when purchasing the medicine they wanted. The factors associated with increased RSM and ISM practice were male gender and not having health insurance. In addition, living in the Highlands was associated with RSM. On the other hand, being 40 to 59 years old or 60 to older were associated with a lower frequency of RSM and ISM.\n\nOur study showed that the prevalence of self-medication was 74.8%, which was higher than that reported by Faria-Domingues et al.23 in a systematic review that aimed to assess the prevalence of self-medication in adult population from Brazil. This review found that one-third of the population self-medicates. Likewise, the prevalence in our study was higher than that found by Jerez-Roig et al.24 in a systematic review that included 28 studies predominantly from Brazil and the United States. Such review showed that the prevalence of self-medication in individuals aged 60 or older was on average 38%, and ranged from 4 to 87%. In our study, the participants were over 18 years old, this may explain the above-average prevalence mentioned in the systematic review of Faria-Domingues et al.23.\n\nEvidence from previous studies shows that self-medication frequency is higher in low and middle-income countries than in developed countries20. Variable prevalence rates have been reported in Latin America, Africa and Asia (27%-86.4%)4,5,9–13,25. However, percentages ranging from 8% to 14%1 are reported in developed countries (United Kingdom, Italy, Switzerland, Belgium, Germany, France, United States of America and the United Kingdom). This is due to the fact that in these countries there is an adequate supervision when supplying OTC drugs. Therefore, most of these prevalence’s correspond to the purchase of OTC medicines. It has also been reported that certain types of drugs such as antibiotics and NSAIDs are available as OTC drugs, causing adverse reactions due to misuse1,16,19,20.\n\nThe most requested drugs by the participants were NSAIDs, antibiotics and analgesics/antipyretics/corticoids. A previous study in Peru found that NSAIDs were also the most acquired drugs (30%)14. On the other hand, in a study carried out in Colombia, the most commonly used drugs were analgesics/antipyretics (44.3%), NSAIDs (36.4%) and antihistamines (8.5%), which goes according to our findings10. These findings are similar because analgesics/antipyretics/corticoids, NSAIDs, antihistamines, and antibiotics are the most frequently used drugs described in other studies7,9,26–31 and are used to treat common symptoms that people do not consider sufficient reason to see a doctor; therefore, they tend to self-medicate.\n\nOur study showed that the male gender reported a higher self-medication frequency. However, studies such as those of Jerez-Roig et al.24 and Lukovic et al.32 reported that the female gender is associated with a higher prevalence of self-medication practice. Our findings are further supported by the study of Quédraogo et al.33, who described that the self-medication practice was found to be associated mostly with the male gender in rheumatic diseases. However, this study was only carried out in an urban area; therefore, the results could vary or not be extrapolated at rural or national level, as is the case of our research.\n\nIn Peru, a study conducted in a district of Metropolitan Lima by Hermoza-Moquillaza et al.14 described that self-medication percentage was higher in the male gender. However, this study did not carry out regression models with multiple variables, which would give our study an innovative character because it is a population-based study and includes a multivariate analysis. On the other hand, findings in Peru regarding a lower self-medication prevalence in females can probably be explained by the sexist nature of its society34. We consider that, in sexist societies, the family structure implies that women are relegated to housekeeping and taking care of children, which, together with the repression from their partner, would reduce their probability to self-medicate and even access to health services. This association could also be explained because men would spend most of their time at work and would not have enough time to go to a health center, so they would resort to the practice of self-medication.\n\nA higher self-medication prevalence was found in participants without health insurance compared to those with SIS. Not having health insurance prevents patient from accessing health services, with the option of self-medicate in drugstores and pharmacies. In this context, some studies have associated the difficulty in obtaining a medical appointment with the self-medication practice, as well as an adverse financial situation35–38. However, in some health systems, having a health insurance does not guarantee a lower self-medication prevalence. Thus, no statistically significant differences were found between people who have SIS or EsSalud and the practice of RSM or ISM. This situation could be explained by the fact that those with SIS can easily make an appointment as an outpatient, but they would not have an adequate access to medicines21. In contrast, the situation with social security is the opposite, people with this insurance can benefit from the adequate supply of medicines under this contributory system. However, making an appointment as an outpatient would represent a more problematic situation compared to people with SIS39. Both situations would eventually lead to RSM or ISM. A similar case occurs in China, where long waiting times (more than half a day), and an expensive medical care, would lead to a high self-medication prevalence of antibiotics in college students40. Similarly, in Saudi Arabia, there was a positive association between the difficult access to health services and the self-medication practice of patients in primary care centers41.\n\nPeru has a fragmented health system, which is divided into two sectors: public and private42,43. The public sector is also divided into subsidized or indirect contributory system and direct contributory or social security system. In the public sector, the government provides medical services (SIS) to the population living in poverty through the MINSA-GR establishments. The social security system is intended for citizens with formal employment. It has two subsystems: EsSalud and the private health care providers. Furthermore, the FF.AA.PP have their own health subsystem. Finally, the private sector is divided into the for-profit system (private insurance companies, private clinics) and non-profit system (NGOs)42,43. The process of universal health insurance in Peru has begun since 2009 and seeks to ensure that more Peruvians have medical insurance based on an essential plan. However, this process is still being implemented and many citizens do not have health insurance yet. This leads to a lack of access to medical services and a high prevalence of ISM in Peru44,45.\n\nWe found an association between the lack of request for prescription by the pharmacist when purchasing the medicine and self-medication in the bivariate analysis. This situation is evidenced by the inadequate distribution of prescription medicines, as is the case with the OTC sale of antibiotics in Peru, despite the current regulations14,46. This situation also occurs in other Latin-American countries such as Chile and Colombia, where there are regulations to prevent the free distribution of antibiotics; however, the results are not evidenced over the years26,47. We also observed a small percentage of participants (6%) who were asked for a prescription despite purchasing an OTC medicine. This would reflect a professional malpractice by pharmacists in Peru.\n\nThis study has limitations: 1) since it is a secondary analysis of a survey designed to assess user’s satisfaction of health services, it was not necessarily conducted to answer our research question; however, the questionnaire has been designed and validated by the INEI staff; 2) the cross-sectional design of this study does not allow us to establish a causal relationship among the factors associated with self-medication. However, it allows us to find the association and identify the markers that could be used by healthcare managers to carry out future public health interventions48.\n\nIn conclusion, there is a high self-medication frequency in Peru, mostly with medicines that are not authorized for OTC sale. It is important to carry out public health interventions in order to reduce the ISM frequency in Peru. There is also a need for educational reforms aimed at raising awareness of the consequences of this irresponsible practice. Similarly, respective measures should be taken to improve the coverage of universal health insurance, thus preventing people from resorting to ISM due to lack of access to medical services. Finally, efforts should be made to integrate druggists and pharmacists into regulatory entities in order to control OTC and uncontrolled sales of prescription drugs. Self-medication could represent a quick and economical solution for users, but it must be practiced within a responsible context.\n\n\nData availability\n\nData associated with this study is available at National Superintendency of Health (Superintendencia Nacional de Salud, SUSALUD) website: http://portal.susalud.gob.pe/blog/base-de-datos-2015/\n\nQuestionnaire analyzed is available online: http://portal.susalud.gob.pe/wp-content/uploads/archivo/encuesta-sat-nac/2015/Cuestionario-4-DIRIGIDA-A-USUARIOS-DE-FARMACIAS-BOTICAS.pdf", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBennadi D: Self-medication: A current challenge. J Basic Clin Pharm. 2014; 5(1): 19–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrganization WH: The Role of the pharmacist in self-care and self-medication: report of the 4th WHO Consultative Group on the Role of the Pharmacist. The Hague, The Netherlands, 26–28 August 1998. 1998. Reference Source\n\nWazaify M, Shields E, Hughes CM, et al.: Societal perspectives on over-the-counter (OTC) medicines. Fam Pract. 2005; 22(2): 170–6. PubMed Abstract \| Publisher Full Text\n\nBalamurugan E, Ganesh K: Prevalence and pattern of self medication use in coastal regions of South India. Br J Med Pract. 2011; 4(3): a428. Reference Source\n\nJalilian F, Hazavehei SM, Vahidinia AA, et al.: Prevalence and related factors for choosing self-medication among pharmacies visitors based on health belief model in Hamadan Province, west of Iran. J Res Health Sci. 2013; 13(1): 81–5. PubMed Abstract\n\nJiménez Rubio D, Hernández Quevedo C: [Differences in self-medication in the adult population in Spain according to country of origin]. Gac Sanit. 2010; 24(2): 116. e1–116.e8. PubMed Abstract \| Publisher Full Text\n\nGuillem Sáiz P, Francès Bozal F, Gimenez Fernández F, et al.: Estudio sobre Automedicación en Población Universitaria Española. Revista Clí nica de Medicina de Familia. 2010; 3(2): 99–103. Publisher Full Text\n\nVacas Rodilla E, Castellà Dagà I, Sánchez Giralt M, et al.: \"[Self-medication and the elderly. The reality of the home medicine cabinet]. Aten Primaria. 2009; 41(5): 269–74. PubMed Abstract \| Publisher Full Text\n\nPeñuela M, dela Espriella A, Escobar E, et al.: Factores socioeconómicos y culturales asociados a la autoformulación en expendios de medicamentos en la ciudad de Barranquilla. Salud Uninorte. 2002; 16: 30–38. Reference Source\n\nMachado-Alba JE, Echeverri-Cataño LF, Londoño-Builes MJ, et al.: Social, cultural and economic factors associated with self-medication. Biomedica. 2014; 34(4): 580–8. PubMed Abstract\n\nLópez JJ, Dennis R, Moscoso SM: [A study of self-medication in a neighborhood in Bogotá]. Rev Salud Publica (Bogota). 2009; 11(3): 432–42. PubMed Abstract\n\nSchmid B, Bernal R, Silva NN: Automedicação em adultos de baixa renda no município de São Paulo. Rev Saude Publica. 2010; 44(6): 1039–45. Publisher Full Text\n\nCorrêa da Silva MG, Soares MC, Muccillo-Baisch AL: Self-medication in university students from the city of Rio Grande, Brazil. BMC Public Health. 2012; 12(1): 339. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHermoza-Moquillaza R, Loza-Munarriz C, Rodríguez-Hurtado D, et al.: Self-medication en un distrito de Metropolitan Area of Lima, Perú. Revista Medica Herediana. 2016; 27(1): 15–21. Reference Source\n\nHughes CM, McElnay JC, Fleming GF: Benefits and risks of self medication. Drug Saf. 2001; 24(14): 1027–37. PubMed Abstract \| Publisher Full Text\n\nMontastruc JL, Bondon-Guitton E, Abadie D, et al.: Pharmacovigilance, risks and adverse effects of self-medication. Therapie. 2016; 71(2): 257–62. PubMed Abstract \| Publisher Full Text\n\nJames LP, Mayeux PR, Hinson JA: Acetaminophen-induced hepatotoxicity. Drug Metab Dispos. 2003; 31(12): 1499–506. PubMed Abstract \| Publisher Full Text\n\nOcan M, Obuku EA, Bwanga F, et al.: Household antimicrobial self-medication: a systematic review and meta-analysis of the burden, risk factors and outcomes in developing countries. BMC Public Health. 2015; 15(1): 742. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSherazi BA, Mahmood KT, Amin F, et al.: Prevalence and Measure of Self Medication: A Review. J Pharm Sci & Res. 2012; 4(3): 1774–1778. Reference Source\n\nShaghaghi A, Asadi M, Allahverdipour H: Predictors of Self-Medication Behavior: A Systematic Review. Iran J Public Health. 2014; 43(2): 136–46. PubMed Abstract \| Free Full Text\n\nMezones-Holguín E, Solis-Cóndor R, Benites-Zapata VA, et al.: Diferencias institucionales en el insuficiente acceso efectivo a medicamentos prescritos en instituciones prestadoras de servicios de salud en Perú: Análisis de la Encuesta Nacional de Satisfacción de Usuarios de los Servicios de Salud (ENSUSALUD 2014). Rev Peru Med Exp Salud Publica. 2016; 33(2): 205–14. Publisher Full Text\n\nRabe‐Hesketh S, Skrondal A: Multilevel modelling of complex survey data. J R Statist Soc A. 2006; 169(4): 805–27. Publisher Full Text\n\nDomingues PH, Galvão TF, Andrade KR, et al.: Prevalence of self-medication in the adult population of Brazil: a systematic review. Rev Saude Publica. 2015; 49: 36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJerez-Roig J, Medeiros LF, Silva VA, et al.: Prevalence of self-medication and associated factors in an elderly population: a systematic review. Drugs Aging. 2014; 31(12): 883–96. PubMed Abstract \| Publisher Full Text\n\nDonkor ES, Tetteh-Quarcoo PB, Nartey P, et al.: Self-medication practices with antibiotics among tertiary level students in Accra, Ghana: a cross-sectional study. Int J Environ Res Public Health. 2012; 9(10): 3519–29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVacca CP, Niño CY, Reveiz L: [Restriction of antibiotic sales in pharmacies in Bogotá, Colombia: a descriptive study]. Rev Panam Salud Publica. 2011; 30(6): 586–91. PubMed Abstract\n\nBertoldi AD, Silveira MP, Menezes AM, et al.: Tracking of medicine use and self-medication from infancy to adolescence: 1993 Pelotas (Brazil) birth cohort study. J Adolesc Health. 2012; 51(6 Suppl): S11–S5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrigoryan L, Burgerhof JG, Degener JE, et al.: Determinants of self-medication with antibiotics in Europe: the impact of beliefs, country wealth and the healthcare system. J Antimicrob Chemother. 2008; 61(5): 1172–9. PubMed Abstract \| Publisher Full Text\n\nGrigoryan L, Haaijer-Ruskamp FM, Burgerhof JG, et al.: Self-medication with antimicrobial drugs in Europe. Emerg Infect Dis. 2006; 12(3): 452–9. PubMed Abstract \| Free Full Text\n\nOrganization WH: Responsible self-care and self-medication; A worldwide review of consumers’ survey. The World Self-Medication Industry. 2010. Reference Source\n\nLam A, Bradley G: Use of self-prescribed nonprescription medications and dietary supplements among assisted living facility residents. J Am Pharm Assoc (2003). 2006; 46(5): 574–81. PubMed Abstract \| Publisher Full Text\n\nLukovic JA, Miletic V, Pekmezovic T, et al.: Self-medication practices and risk factors for self-medication among medical students in Belgrade, Serbia. PLoS One. 2014; 9(12): e114644. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOuédraogo DD, Zabsonré/Tiendrebeogo JW, Zongo E, et al.: Prevalence and factors associated with self-medication in rheumatology in Sub-Saharan Africa. Eur J Rheumatol. 2015; 2(2): 52–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFlake DF: Individual, family, and community risk markers for domestic violence in Peru. Violence Against Women. 2005; 11(3): 353–73. PubMed Abstract \| Publisher Full Text\n\nMontastruc JL, Bagheri H, Geraud T, et al.: [Pharmacovigilance of self-medication]. Therapie. 1996; 52(2): 105–10. PubMed Abstract\n\nGiroud JP, Cupillard C, Curé O: Médicaments sans ordonnance: les bons et les mauvais!. La Martinière; 2011. Reference Source\n\nFainzang S: L'automédication ou les mirages de l'autonomie. Presses universitaires de France. 2015. Reference Source\n\nFainzang S: The other side of medicalization: self-medicalization and self-medication. Cult Med Psychiatry. 2013; 37(3): 488–504. PubMed Abstract \| Publisher Full Text\n\nSánchez-Moreno F: [Inequity in health affects the development in Peru]. Rev Peru Med Exp Salud Publica. 2013; 30(4): 676–82. PubMed Abstract\n\nZhu X, Pan H, Yang Z, et al.: Self-medication practices with antibiotics among Chinese university students. Public Health. 2016; 130: 78–83. PubMed Abstract \| Publisher Full Text\n\nAlghanim SA: Self-medication practice among patients in a public health care system. East Mediterr Health J. 2011; 17(5): 409–16. PubMed Abstract \| Publisher Full Text\n\nSánchez-Moreno F: [The national health system in Peru]. Rev Peru Med Exp Salud Publica. 2014; 31(4): 747–53. PubMed Abstract\n\nAlcalde-Rabanal JE, Lazo-González O, Nigenda G: [The health system of Peru]. Salud Publica Mex. 2011; 53 Suppl 2: s243–s54. PubMed Abstract\n\nEibenschutz C, Valdivia AS, González ST, et al.: Considerations on health reform process (1993-2013) and social participation in Peru. Saúde em Debate. 2014; 38(103): 872–82. Publisher Full Text\n\nWilson L, Velásquez A, Ponce C: La ley marco de aseguramiento universal en salud en el Perú: análisis de beneficios y sistematización del proceso desde su concepción hasta su promulgación. Rev Peru Med Exp Salud Publica. 2009; 26(2): 207–17. Reference Source\n\nEcker L, Ruiz J, Vargas M, et al.: [Prevalence of purchase of antibiotics without prescription and antibiotic recommendation practices for children under five years of age in private pharmacies in peri-urban areas of Lima, Peru]. Rev Peru Med Exp Salud Publica. 2016; 33(2): 215–23. PubMed Abstract \| Publisher Full Text\n\nBavestrello FL, Cabello MA: [Community antibiotic consumption in Chile, 2000-2008]. Rev Chilena Infectol. 2011; 28(2): 107–12. PubMed Abstract \| Publisher Full Text\n\nRothman KJ, Gallacher JE, Hatch EE: Why representativeness should be avoided. Int J Epidemiol. 2013; 42(4): 1012–4. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "42722", "date": "23 Jan 2019", "name": "Marcus Tolentino Silva", "expertise": [ "Reviewer Expertise epidemiology", "health technology assessment", "pharmacy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a national survey in Peru performed in drugstores and pharmacies in 2015. The authors stratified their respondents in: (i) non self-medication (NSM, purchased based a prescription); (ii) responsible self-medication (RSM, paid an OTC); or (iii) irresponsible self-medication (IRM, acquire a prescription drug without prescription). The results reveal that RSM and IRM are more frequent in males and without health insurance, and more uncommon in >39 years old.\nSome suggestions for improvement:\nIn Table 1, please stratify the characteristics of participants by the three response variables (NSM, RSM, IRM) and exclude the Table 2 (male data are missing). Change the Figure 2 to table for better visualization.\n\nI believe that the results are influenced by the NSM group. This population had a prescription, which is, visited more health services and probably had poor health status. Such characteristics are attributable to women and elders. The poor health status of the NSM group induced their best behavior. Sexism is important, but I think that it is not sufficient to explain worse male health behavior. Please review these aspects in the discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5158", "date": "24 Jan 2020", "name": "Diego Urrunaga-Pastor", "role": "Author Response", "response": "We have read the reviewers commentaries and want to clarify certain points in this new version: Dear Dr. Marcus Tolentino-Silva: 1. In Table 1, please stratify the characteristics of participants by the three response variables (NSM, RSM, IRM) Answer: Thank you very much for the commentary, however, we consider important to keep table 1 with the descriptive analysis and to present the 3 strata of our main variable (NSM, RSM, ISM) in table 2. 2. Exclude the Table 2 (male data are missing) Answer: Thank you for the observation, we have added the missing data in table 3. In addition, we have presented the bivariate analysis based on the 3 strata of the main variable. 3. Change the Figure 2 to table for better visualization. Answer: We appreciate the comment. We have removed figure 2 and replaced it by a table (table 2) for better visualization. 4. I believe that the results are influenced by the NSM group. This population had a prescription, which is, visited more health services and probably had poor health status. Such characteristics are attributable to women and elders. The poor health status of the NSM group induced their best behavior Answer: Thank you for the commentary, we have reviewed the literature, expanding this discussion section with this information. 5. Sexism is important, but I think that it is not sufficient to explain worse male health behavior. Please review these aspects in the discussion. Answer: Thank you for the commentary, we did a review of the literature regarding this topic, expanding the information presented in the discussion." } ] }, { "id": "42721", "date": "28 May 2019", "name": "Cristian Diaz Veliz", "expertise": [ "Reviewer Expertise Médico Epidemiologo", "Oficina de Inteligencia Sanitaria del Hospital Nacional Almanzor Aguinaga Asenjo" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe sample calculation mentions the use of 30% satisfaction, but it is not a used variable. The first paragraph of the discussion should go into the results section. It shows results of types of drugs most used in self-medication, but in the discussion this is not mentioned. See existing studies published in Peru (Lima and Lambayeque). Another limitation of the study not mentioned, is the effect of the pharmacies in the dispensing of medicines, when changing or indicating drugs. See Study: Alterations in drug dispensation by private sector pharmacies in the district of Chiclayo1.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5160", "date": "24 Jan 2020", "name": "Diego Urrunaga-Pastor", "role": "Author Response", "response": "We have read the reviewers commentaries and want to clarify certain points in this new version: Dear Dr. Cristian Díaz-Vélez: 1. The sample calculation mentions the use of 30% satisfaction, but it is not a used variable. Answer: Thank you for the observation, ENSUSALUD was developed to evaluate health user satisfaction, therefore the INEI did not perform a sample size calculation based on self-medication prevalence. 2. The first paragraph of the discussion should go into the results section. Answer: Thank you for the suggestion, however, we consider keeping the paragraph as a summary of our main findings. 3. It shows results of types of drugs most used in self-medication, but in the discussion, this is not mentioned. See existing studies published in Peru (Lima and Lambayeque). Answer: Thank you for the commentary, in the 4th paragraph of the discussion section we have cited international and national studies to mention these results. 4. Another limitation of the study not mentioned is the effect of the pharmacies in the dispensing of medicines when changing or indicating drugs. See Study: Alterations in drug dispensation by private sector pharmacies in the district of Chiclayo1. Answer: Thank you for the suggestion, however, it is not part of the objectives of our study and we did not have an instrument to measure this variable." } ] } ]	1	https://f1000research.com/articles/8-23
https://f1000research.com/articles/8-1949/v1	21 Nov 19	{ "type": "Research Article", "title": "Social and cultural factors perpetuating early marriage in rural Gambia: an exploratory mixed methods study", "authors": [ "Mat Lowe", "Mamsamba Joof", "Bomar Mendez Rojas", "Mamsamba Joof", "Bomar Mendez Rojas" ], "abstract": "Background: Over the last two decades, early marriage in the Gambia declined significantly (from 58% to 30%). Yet evidence indicates that nearly 8.6% of marriages in the Gambia involved girls younger than 15, and 46.5% of marriages are with girls aged 18 or below. The reasons for the decline but continuing practice of early marriage, despite existing legislation prohibiting child marriage, are not very well understood. Very few studies have been conducted to find out what and how local factors influence decisions about early marriage in the Gambia. More information is therefore needed on underlying reasons for the persistence of early marriage in the Gambia so that program managers can use this information to design strategies towards accelerating the decline of early marriages. Methods: The study was conducted in 24 rural settlements in Lower Baddibu District in the North Bank Region of the Gambia. It was based on a mixed-methods design including a cross-sectional household survey with a sample of 181 female adolescents and focus group discussions with 16 male and female parents. Focus group discussions were digitally-recorded, transcribed verbatim and analyzed using thematic content analysis, while survey data were analyzed using Stata. Results: Using multiple regression analysis, this study found that ethnicity more than other factors, exerts an independent effect on early marriage. Themes identified during focus group discussions also revealed that fear of premarital sex and loss of virginity outside marriage were major reasons for the perpetuation of early marriage. Conclusions: These findings suggest that the practice of early marriage in rural Gambia is associated with ethnicity and practices related to social and cultural norms. The findings also suggest that in order to decrease early marriages, future efforts should focus on allaying the fears around premarital sex and loss of virginity related to delay in marriage.", "keywords": [ "Early marriage", "Gambia", "mixed-methods", "female adolescents", "average age", "prevalence", "virginity", "premarital" ], "content": "Introduction\n\nEarly marriage or child marriage, defined as marriage before the age of 18, is perceived as a grave violation of human rights. It is a global problem, even though it is prohibited by the Convention on the Rights of the Child and the Convention on the Elimination of All Forms of Discrimination against Women37,38, two of the most broadly endorsed international human rights agreements. In the last decade, the practice of early marriage declined worldwide from 25% to 21%, a modest reduction of 15%1.\n\nHowever, the rate of decline has been slowest in West and Central Africa, the region with the highest prevalence of child marriage1,2. Within the region, estimates vary from 76% in Niger to 18% in Cape Verde3. In West and Central Africa, four in ten girls4 marry before the age of 18, and one in three marries before age 15. At this rate, with the growing population of girls in the region, the number of child brides in West and Central Africa is projected to increase from 6.4 million in 2015 to 7.1 million by 20302.\n\nThe reasons why early marriage is so common in West and Central Africa are wide ranging and can be grouped under religion, tradition and culture, poverty, and gender inequalities8,20,21,36. However, there is evidence that research findings from communities cannot be generalized to other countries or even to other communities within the country where the research was conducted22. Rather, because of this inability to generalize it is crucial to use locally derived evidence to understand how and why communities differ in their approach to child marriage so that more sensitive strategies can be developed to address the problem. The purpose of this study, therefore, was to provide a contextualized picture of underlying factors that are perpetuating early marriage in rural Gambian communities.\n\n\nEarly marriage in the Gambia\n\nA National Child Protection Strategy Plan, which highlighted early marriage as an issue to address, was launched in 2016. Despite this, early marriages are still common in the Gambia which is listed in the top 41 countries worldwide where the prevalence of child marriage is 30% or more44. Historically, early marriages were widespread with 58% of 40–49-year-old marrying before the age of 18. But this declined over time and now an estimated 30 percent of women aged 20–24 marry before the age of 18. The reasons for this decline are uncertain although increasing enrolment of girls in school may be a contributory factor43.\n\nGirls are more affected than boys, with a girl-to-boy ratio for marriage before the age of 18 of 43:125. Girls most at risk come from poorer rural households with little or no education25,44. Urban women aged 25–49 tend to marry about two years later than their rural counterparts. For instance, women in Banjul the capital city marry four years later than women in Kuntaur, a rural settlement 21.0 versus 17.0. For women who have secondary or higher education the median age to marry is 22.2 compared to 17.3 for women who have no education. Similarly, women in the wealthiest quintile marry at a median age of 20.8 years whereas women in the lowest quintiles marry at 17.2 years25. Adolescents are aware of contraceptives (91%) but rarely use them (3.3%), so that childbearing begins early in the Gambia25. Almost one in five (18 percent) of adolescent women age 15–19 are already mothers or pregnant with their first child25.\n\nThe reasons for why the practice of early marriage still persists in the Gambia despite the initial decline and the effect of the recent 2016 legal reforms to outlaw early marriage45 have received inadequate attention aside from a single published report34. More information is therefore needed on underlying reasons for the persistence of early marriages in the Gambia. But even more importantly no studies have yet been conducted to design interventions based on locally generated research findings that fully characterize contextual factors influencing decisions on early marriage in rural Gambian communities. The primary purpose of this study is to address these gaps and to provide program managers with detailed information on the type and short-term effects of contextually relevant interventions that can be scaled up to change societal attitudes towards accelerating the decline of early marriages in rural Gambia. The study was conducted as part of a larger research project addressing teen pregnancy and early marriage in the Gambia41.\n\n\nMethods\n\nThis study was based on a mixed-methods design that included a cross-sectional household survey and focus group discussions as primary data collection techniques. It was conducted in 24 rural settlements in Lower Baddibu District in the North Bank Region of the Gambia. Lower Baddibu District has a population of 17,96124. It had the second-lowest recorded median age at marriage (17.3 years) in a national survey25.\n\nCross-sectional household survey. The sampling frame for the cross-sectional household survey was drawn from the Directory of Settlement24, which is the census frame. The sample was selected in two stages. First, all the 24 settlements in Lower Baddibu District were randomly selected and grouped under the four main ethnic groups, Mandinka, Fula, Wolof and Serer, with probability proportional to population size age 10 years and above for each ethnic group of settlement (Table 1). Further stratification by age was done. Those to be interviewed were divided into three age groups: a) 10–19 years; b) 20–39 years; and c) 40+ years. The proportion of the population in the Gambia that falls into each of these groups is estimated at 35% for 10–19; 42% 20–39 and 23% for those aged 40+24. These proportions were used to determine the number of those to be interviewed within each age group in each ethnic group of settlements (Table 2). Female adolescents represent 56% (181 out of 320 adolescents) of the total 10–19 years targeted for the survey (Table 2). Female adolescents (aged 10–19 years) were the main focus of the survey because the practice of early marriage has a greater impact towards young females.\n\nWhen the research team arrived in the selected settlement, a systematic sampling approach was used to select every second household, followed by convenience sampling to select respondents within each household. Two female respondents aged 10- to 19-year were selected. The research team then continued to collect data in the settlement until they obtained the numbers required. However, if the households did not provide the number of respondents required, then the research team subsequently moved to a random new settlement. Interviews were conducted at noon and sometimes in the late evening to maximize the response rate. No more than two individuals per household in the same age group were interviewed to increase representativeness.\n\nFocus group discussions. The participants were selected purposively, and were mainly parents, who were recruited voluntarily with the help of the research team. They were chosen based on their role as household heads with actual or potential relevance and understanding of decision-making processes around early marriage.\n\nQuantitative data. Two types of questionnaires (a Household Questionnaire and a Female Adolescent Questionnaire, both available as Extended data49,50) were used to conduct face to face interviews with interviewees. The Household Questionnaire was used to identify female adolescents eligible to be interviewed with the Female Adolescent Questionnaire. In addition, the Household Questionnaire also collected information on the condition of the dwelling and household amenities and possessions, such as the source of drinking water, type of sanitation facilities, materials used to construct the dwelling, and ownership of various consumer durables, land, and farm animals. The Female Adolescent Questionnaire collected information on characteristics of female adolescents (age, marital status, ethnicity, literacy, current school attendance, education, employment, exposure to early marriage prevention messages, average age of marriage and characteristics of parents). The questionnaires were adapted from a similar study in Ethiopia26 and from the Demographic Health Survey of the Gambia25. They were pre-tested in three settlements in Sabach Sanjal District, which have similar characteristics to Lower Baddibu District, where the study was conducted. The pre-testing allowed revisions and finalization of the questionnaires before they were put to full-scale administration.\n\nQualitative data. For the qualitative data collection, two focus group discussions that included 16 participants including both male and female parents were conducted in two selected case study settlements (Njwara and Foday Biran). The focus group discussions typically lasted for 45 minutes and were held in either the village health post or community center to avoid distraction. They were conducted in the vernacular languages (Mandinka, Wolof, Fula and Serer) by the main author and the research team, which comprised of four data collectors and two field supervisors. The main author and the research team have a thorough mastery of the four main languages (Mandinka, Wolof, Fula and Serer) used to conduct the focus group discussions. A focus group discussion guide which was developed based on review of existing literature was used to facilitate discussion during focus group discussions (this is available as Extended data48). The focus group discussion guide explored perceptions of and attitudes towards early marriage. It was pre-tested with participants that have similar inclusion criteria as those who participated in the focus group discussions. Focus group discussions were digitally recorded using an IC recorder and transcribed verbatim in English language.\n\nQuantitative data. Descriptive analysis of survey respondents’ demographic characteristics was first conducted using the means and standard deviations for continuous variables and the frequencies and percentages for categorical variables. Second, multiple logistic regression analysis was used to determine the predictors of early marriage among female adolescents. We used a progressive analytical strategy starting with the calculations of the unadjusted odds ratios. We first ran model 1 in which ethnic groups were adjusted by messages to prevent early marriage. In model 2 we assessed whether the observed associations of model 1 were explained by the presence of selected household characteristics (a proxy of wealth). In models 3 and 4, we evaluated the extent the associations of previous model are explained by female´s education, adolescent parents’ survival status, adolescent`s parent ability to read and write and whether the female adolescent agree that fathers as heads of household and not mothers should arrange for their daughters’ and sons’ marriage. All the analyses were conducted in Stata version 12.0 produced by StataCorp in College Station, TX.\n\nQualitative data. Data analysis process for the qualitative data involved listening to all sound recording files from focus group discussions by the main author and the research team before transcription began. After transcription, the transcribed text was read at least three times to make sense of totality. This process of transcription and reading of the transcribed texts allowed identification of thematic categories, which were later developed into major themes. Coding and analysis of all data were subjected to content analysis28, and NVivo 11 Pro was used to manage the data.\n\nBefore the start of the study, the main author submitted the study proposal and tools to the Scientific Coordinating Committee (SCC) Medical Research Council (MRC) The Gambia at the London School of Hygiene and Tropical Medicine (LSHTM). The main author was then invited to present at the meeting of the SCC. The SCC provided some inputs on the study proposal and tools following presentation by the main author at its meeting. The revised study proposal and tools in relation to the issues raised by the SCC was re-submitted by the main author and later approved by the SCC. In the study proposal and tools was later submitted and approved by the SCC (SCC 1651v1.1). The SCC then subsequently forwarded the approved study proposal and tools to the Joint Gambia Government/MRC Ethics Committee for further consideration, which later provided ethical approval (SCC 1651v1.2) following minor changes on the informed consent form.\n\nDuring data collection, informed consent, verbal and written depending on the level of literacy, were obtained from the study participants. Parental informed consent was sought for minors under the age of 18 years and since the research team were composed of men, permission was also taken from husbands of married female adolescents because husbands can decide whether their women should talk to other men or not42.\n\nCare was also taken to make sure that all questions were asked in a culturally respectful and non-judgmental way. This was achieved through the careful selection and training of data collectors and field supervisors, as well as by the design of the study tools. In all the study communities, courtesy calls were also made to village heads (locally known as “Alkaloes”) to inform them about the purpose of the study and to seek clearance from them. Although this method of obtaining approval is considered customary, it is highly ethical and recommended, since village heads yield much power over their people and territories.\n\n\nResults\n\nThe results are presented in two sections. First, data on demographic characteristics of female adolescents are presented, followed by factors associated with early marriage. Individual-level responses to questionnaires47 and de-identified summary transcripts of focus group discussions are available as Underlying data. Focus group transcripts46 are available as Underlying data.\n\nTable 3 presents the demographic characteristics of female adolescents in the cross-sectional household survey. Nearly 70% of female adolescents belonged to the Mandinka and Fula ethnic groups and are older than 13 years. About 12% of them are currently or ever married. On average, female adolescents have 4.2 years of schooling and about 45% are currently attending school. The average age at which female adolescents first heard that their parents had arranged for their marriage was 16 years (Figure 1), which is below the national legal age of 18 years of marriage.\n\nFive villages are not displayed because they have no data on average age at which female adolescent first heard that her parents had arranged her marriage.\n\nThe vertical orange line is the average age at which the female respondent first heard that her parents arranged her marriage. The orange area represents the 95% confidence interval of the average. The average was 16 years, the minimum value of the average was 15.1 years, and the maximum value was 16.6 years. The horizontal red lines and circles represent villages in which the average age is below the minimum value, the blue lines represent the villages in which the average age is above the maximum value, and the green lines represent the villages in which the average age falls within the average range for this area.\n\nFactors identified using multiple logistic regression analysis. Table 4 shows regression analysis results for factors associated with marrying before 18 years among female adolescents. In the unadjusted associations, females in both the Mandinka and Wolof ethnic groups have a higher probability of marrying before 18 years than do females in the Fula ethnic group. Compared with the reference group, females in the Mandinka ethnic group have a 3-fold higher probability of marrying before 18 years, and those in the Wolof ethnic group have a 2.9-fold higher probability; both results are statistically significant at the 0.05 level.\n\n**The results are statistically significant at 0.05 level. 1= Unadjusted odds ratio; 2= The Serer ethnic group has no enough observations to be included in this logistic regression analysis\n\nIn Model 1, we entered the ethnic groups simultaneously and whether the respondent was exposed to messages regarding the prevention of early marriage, as classified by the number of sources (e.g., radio, TV). After the number of messages received was held constant, females in the Mandinka and Wolof ethnic groups had a 2-fold greater probability of marrying early than did females in the Fula ethnic group.\n\nIn Model 2, we evaluated the association of ethnic group and probability of marrying before 18 years while holding constant the socioeconomic condition of the family and the messages of early marriage prevention. As a proxy of family socioeconomic condition, we created a composite index of six household characteristics, where the higher the number of characteristics in the households, the higher the socioeconomic condition. When household characteristics are taken into account in Model 2, the significance level for the Mandinka ethnic group disappeared, while that for the Wolof ethnic group remains statistically significant. The lack of significance for the Mandinka ethnic group indicates that part of the effect observed in Model 1 was explained by the socioeconomic condition of the family and was not an effect of the Mandinka ethnic group by itself. On the other hand, the result of the Wolof ethnic group indicates that independent of the socioeconomic conditions, this ethnic group has a higher probability of marrying early.\n\nIn addition to the variables included in Model 2 and Model 3, we included the females’ duration of schooling. The inclusion of this variable will help to elucidate whether female education may confound the associations among ethnic group, family socioeconomic condition and marrying before 18 years. The results of Model 2 indicate that when family socioeconomic conditions and adolescent female education are held constant, females in the Wolof ethnic group have a 3-fold higher probability of marrying before 18 than do females in the Fula ethnic group.\n\nIn Model 4, we added five more variables to those evaluated in Model 3. The results show that when family socioeconomic condition, adolescent female education, living mother, living father, mother’s/father’s ability to read and write and whether the female adolescent agrees that fathers, as heads of household, and not mothers, should arrange for their daughters’ and sons’ marriage are held constant, females in the Wolof ethnic group still have nearly a 3-fold higher probability of marrying early than females in the Fula ethnic group. All these results indicate that ethnicity, particularly Wolof, exert an independent effect on the probability of marrying early. These results were crossed examined with findings from the focus group discussions, which explored alternative explanations for early marriage.\n\nThemes identified during focus group discussions. Other factors associated with early marriage also included practices related to social and cultural norms. Findings from the focus group discussions revealed that fear of premarital sex and loss of virginity outside marriage were reported as major factors in the perpetuation of early marriage. In one focus group discussion, a participant, was quoted as saying: “It is very risky to delay marriage for your daughter because you don’t know what that might become of her”. In another focus group discussion, a participant, added the following: “In fact, in the first place, there is no guarantee that she [your daughter] is not having love affairs. To be sure that she does not have sexual encounter or loss her virginity before marriage, it is better to marry her off”. From the above statements it is clear that fear of premarital sex is a major factor in early wedlock. It is also clear that ensuring virginity at marriage is highly regarded and therefore fuels the belief that girls should marry early. These issues are further complicated by the perceived lack of good potential husbands and the limited meaningful alternatives to marriage available for girls, such as explained by this participant: “There is great pressure on us [parents] to delay marriage for our girls. We at times submit to this pressure. But the thing is that a good husband is also hard to come by so that when this opportunity arises for your girl, you are left with no other options but to marry her off”.\n\nTaken together, these factors might at least partly explain the reasons for the continuing practice of early marriage in rural Gambian communities in Lower Baddibu District.\n\n\nDiscussion\n\nOur findings suggest that the practice of early marriage in rural Gambia is associated with ethnicity and practices related to social and cultural norms. We found that, ethnicity, particularly Wolof, exerts an independent effect on the probability of marrying early. This finding is consistent with a study31 that showed ethnic factors as determinants of early marriage among young females. Our findings also revealed that among parents, fear of premarital sex and loss of virginity outside marriage were reported as major factors in the perpetuation of early marriage. This finding is in keeping with the chastity explanation as proposed by Bicchieri et al.33, which explains that parents want their daughters to be chaste and believe that there is a risk that girls who grow older may lose their virginity outside of marriage. The finding has wide programmatic implications. It suggests that in order to prevent early marriages, future efforts should focus on allaying the fears around premarital sex and loss of virginity related to delay in marriage. Such efforts may include providing girls with age appropriate information to prevent early sexual activities and engaging parents on discussions focusing on the social norms around early marriage. These strategies are contextually relevant and can be implemented and scaled up to change societal attitudes towards accelerating the decline of early marriages in rural Gambia. For instance, many parents in our focus group discussions supported providing girls with information about the harmful effects of premarital sex and teenage pregnancies, as well as organizing community engagement forums with parents as effective package of interventions that can be implemented to prevent early marriage in rural Gambia.\n\nWhile our study has provided a contextualized picture of the underlying social and cultural factors that are perpetuating early marriage in rural Gambia, the findings should be interpreted in light of the following limitations. First, it was conducted in a single district, which limits generalization to other districts. There is evidence that study findings on early marriage from communities cannot be generalized to other countries or even to other communities within the country where the study was conducted22. Second, because child marriage is illegal in the Gambia, participants may have provided measured responses for fear that they may be blamed by other community members and/or possibly be prosecuted by providing enough information on the situation of early marriage. Finally, although the study has touched on some important factors perpetuating early marriage, the findings are by no means all the social and cultural factors that perpetuate early marriage in rural Gambian communities. Nonetheless, the study has important implications for policy and or practice. It can be used by program managers to accelerate the decline of early marriages in the Gambia.\n\nFor future research, the study indicates the need for a more robust population-based study, which would have the potential to provide a more contextualized picture of the prevalence and factors that are perpetuating early marriages in rural Gambian communities.\n\n\nData availability\n\nFigshare: Social and cultural factors perpetuating early marriage in rural Gambia: an exploratory mixed methods study. https://doi.org/10.6084/m9.figshare.10055516.v146.\n\nThis project contains de-identified summary transcripts of focus group discussions. Focus group transcripts.\n\nFigshare: Social and cultural factors perpetuating early marriage in rural Gambia: an exploratory mixed methods study. https://doi.org/10.6084/m9.figshare.10045979.v147.\n\nThis project contains responses to the questionnaires administered in this study.\n\nFigshare: Social and cultural factors perpetuating early marriage in rural Gambia: an exploratory mixed methods study. https://doi.org/10.6084/m9.figshare.10046024.v148.\n\nThis project contains the focus group discussion guide.\n\nFigshare: Social and cultural factors perpetuating early marriage in rural Gambia: an exploratory mixed methods study. https://doi.org/10.6084/m9.figshare.10046090.v149.\n\nThis project contains the questionnaire administered to the female adolescents.\n\nFigshare: Social and cultural factors perpetuating early marriage in rural Gambia: an exploratory mixed methods study. https://doi.org/10.6084/m9.figshare.10046102.v150.\n\nThis project contains the household questionnaire.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors are grateful to the participants for sharing their insights and to the research team, namely, Mariama Bojang, Alhagie Gaye, Karamo K. Kinteh, Marie Noel Mendy, Momodou A. Jallow, Omar Touray and Sheikh O. Fye, for their tireless efforts to collect the data.\n\n\nReferences\n\nChild marriage: latest trends and future prospects. Reference Source\n\nUnited Nations Population Fund: Marrying too young end child marriage. New York, NY: UNFPA. 2012. Reference Source\n\nUNICEF: Monitoring the status of children and women. 2018; Accessed 8 Aug 2018. Reference Source\n\nUNICEF: Achieving a future without child marriage. Reference Source\n\nPopulation Reference Bureau: The world’s youth 2006 data sheet, bridge. 2006. Reference Source\n\nUnited Nations Children’s Fund: Ending child marriage: progress and prospects. New York, NY: UNICEF. 2014. Reference Source\n\nLarsen JE: Young people in west and Central Africa trends, priorities, investments and partners, UNICEF. 2009; Accessed 10 Aug 2018. Reference Source\n\nChild marriage in West and Central Africa – Girls not Brides, The Global Partnership to end Child Marriage. Reference Source\n\nNour NM: Health consequences of child marriage in Africa. Emerg Infect Dis. 2006; 12(11): 1644–9. PubMed Abstract \| Free Full Text\n\nWorld Health Organization: Report of the expert group meeting on mainstreaming adolescent pregnancy in the work of the World Health Organization. 2009; Accessed 21 Aug 2018. Reference Source\n\nWHO: WHO Guidelines on Preventing Early Pregnancy and Poor Reproductive Health Outcomes Among Adolescents in Developing Countries. Geneva, Switzerland: WHO; 2011. PubMed Abstract\n\nMaheu-Giroux M, Filippi V, Maulet N, et al.: Risk factors for vaginal fistula symptoms in Sub-Saharan Africa: a pooled analysis of national household survey data. BMC Pregnancy Childbirth. 2016; 16: 82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNguyen MC, Wodon Q: Early Marriage, Pregnancies, and the Gender Gap in Education Attainment: An Analysis Based on the Reasons for Dropping out of School. In Child Marriage and Education in sub-Saharan Africa. Wodon Q, Washington DC: World Bank. 2015b; (forthcoming).\n\nParsons J, Edmeades J, Kes A, et al.: Economic impacts of child marriage: a review of the literature. Rev Faith Int Aff. 2015; 13: 12–22. Publisher Full Text\n\nUNICEF: Hidden in plain sight: a statistical analysis of violence against children. New York, NY: UNICEF; 2014. Reference Source\n\nAbu-Ghaida D, Klasen S: The costs of missing the millennium development goal on gender equity. World Dev. 2004; 32(7): 1075–107. Publisher Full Text\n\nEconomic impacts of child marriage: global synthesis report. Reference Source\n\nSmith LC, Haddad L: Reducing child undernutrition: past drivers and priorities for the post-MDG era. World Dev. 2015; 68: 180–204. Publisher Full Text\n\nGirls not brides: Child marriage in West and Central Africa. 2017; Accessed 9 Aug 2018. Reference Source\n\nJohansson N: Child marriage: the underlying reasons and possible solutions. Linnaeus University, Faculty of Social Sciences, Department of Social Studies. 2015. Reference Source\n\nQuentin W, Chata M, Adenike O, et al.: Girls’ education and child marriage in West and Central Africa: key findings ahead of the October 2017 high level meeting on ending child marriage in West and Central Africa (French). Washington, D.C: World Bank Group; 2017. Reference Source\n\nLessons learned from national initiatives to end child marriage. 2016. Reference Source\n\nChant S, Touray I: Gender in The Gambia in retrospect and prospect. GAMCOTRAP Working Paper. 2012. Reference Source\n\n2013 Population and Housing Census, Directory of Settlements, Gambia Bureau of Statistics Banjul, The Gambia.\n\nDemographic and Health Survey: Gambia bureau of statistics banjul. Maryland, USA: The Gambia ICF International Rockville; 2013.\n\nGage A: Coverage and Effects of Child Marriage Prevention Activities in Amhara Region, Ethiopia. 2009. Reference Source\n\nNicola J, Fiona S, Carol W: Question guide: researching norms about early marriage and girls’ education. 2015. Reference Source\n\nBraun V, Clarke V: Using thematic analysis in psychology. Qual Res Psychol. 2006; 3(2): 77-101. Publisher Full Text\n\nMenon JA, Kusanthan T, Mwaba SOC, et al.: 'Ring' your future, without changing diaper - Can preventing teenage pregnancy address child marriage in Zambia? PLoS One. 2018; 13(10): e0205523. Publisher Full Text\n\nThe Gambia-Multiple indicator cluster survey 2010. Fourth Round. Reference Source\n\nRumble L, Peterman A, Irdiana N, et al.: An empirical exploration of female child marriage determinants in Indonesia. BMC Public Health. 2018; 18(1): 407. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nA baseline study on child marriage, teenage pregnancy and female genital mutilation/cutting in Kenya. Reference Source\n\nBicchieri C, Jiang T, Lindemans JW: A social norms perspective on child marriage: The general framework. Penn Soc Norms Group (Pennsong). 2014; 13, Accessed January 29, 2019. Reference Source\n\nJouhk J, Stark L: Causes and motives of early marriage in the Gambia and Tanzania. Reference Source\n\nGhaffari M, Gharlipour Gharghani Z, Mehrabi Y, et al.: Premarital Sexual Intercourse-Related Individual Factors Among Iranian Adolescents: A Qualitative Study. Iran Red Crescent Med J. 2016; 18(2): e21220. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNour NM: Health Consequences of Child Marriage in Africa. Emer Infect Dis. 2006; 12(11): 1644–1649. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUNG Assembly-United Nations, Treaty Series, 1989-wunrn. Reference Source\n\nhttp://www.bayefsky.com/general/cedaw_c_2006_ii_4.pdf\n\nThe Research Ethics Guide Book. Reference Source\n\nA guide to developing end child marriage projects and how to fund raise them. Reference Source\n\nAddressing teen pregnancy and early marriage in the Gambia. Reference Source\n\nLowe M, Chen DR, Huang SL: Social and Cultural Factors Affecting Maternal Health in Rural Gambia: An Exploratory Qualitative Study. PLoS One. 2016; 11(9): e0163653. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJouhki J, Stark L: Causes and Motives of Early Marriage in The Gambia and Tanzania. Is New Legislation Enough? University of Jyväskylä, Poverty and Development Working Papers, 1/2017. Reference Source\n\nLessons learned from national initiatives to end child marriage –Girls not Brides. The Global Partnership to end Child Marriage. 2016. Reference Source\n\nUNICEF: A Big Win for Girls on the Banning of Child Marriage in The Gambia, 2016a. Reference Source\n\nLowe M, Rojas BM, Joof M: Social and cultural factors perpetuating early marriage in rural Gambia: an exploratory mixed methods study. figshare. Journal contribution. 2019. http://www.doi.org/10.6084/m9.figshare.10055516.v1\n\nLowe M, Rojas BM, Joof M: Social and cultural factors perpetuating early marriage in rural Gambia: an exploratory mixed methods study. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.10045979.v1\n\nLowe M, Rojas BM, Joof M: Social and cultural factors perpetuating early marriage in rural Gambia: an exploratory mixed methods study. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.10046024.v1\n\nLowe M, Rojas BM, Joof M: Social and cultural factors perpetuating early marriage in rural Gambia: an exploratory mixed methods study. figshare. Journal contribution. 2019. http://www.doi.org/10.6084/m9.figshare.10046090.v1\n\nLowe M, Rojas BM, Joof M: Social and cultural factors perpetuating early marriage in rural Gambia: an exploratory mixed methods study. figshare. Journal contribution. 2019. http://www.doi.org/10.6084/m9.figshare.10046102.v1" }	[ { "id": "56901", "date": "17 Dec 2019", "name": "Oluwaseyi Abiodun Akpor", "expertise": [ "Reviewer Expertise adolescent and maternal health" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study was aimed at providing information on the type and short-term effects of contextually relevant interventions that can be scaled up to change societal attitudes towards accelerating the decline of early marriages in rural Gambia. Overall, the study design was appropriate with robust data collection and interpretation. The data were adequately interpreted and discussed.\n\nThe authors should however reduce the length of the background in the abstract and include more findings in the result section.\n\nThe manuscript is recommended for indexing, subject to the minor revision.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "57612", "date": "24 Dec 2019", "name": "Peter J. Winch", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract:\nConclusions repetitive of the Results section of the Abstract.\n\nResults and Conclusions sections not well integrated, and are not very specific. What about ethnicity factors early marriage? What are the social and cultural norms? Much repetition between the two sections of the Abstract. Does not create excitement in the reader to download and read this paper.\n\nResults: Using multiple regression analysis, this study found that ethnicity more than other factors, exerts an independent effect on early marriage. Themes identified during focus group discussions also revealed that fear of premarital sex and loss of virginity outside marriage were major reasons for the perpetuation of early marriage.\n\nConclusions: These findings suggest that the practice of early marriage in rural Gambia is associated with ethnicity and practices related to social and cultural norms. The findings also suggest that in order to decrease early marriages, future efforts should focus on allaying the fears around premarital sex and loss of virginity related to delay in marriage.\n\nIntroduction:\nGood that they presented a universal definition of child marriage at the very beginning.\n\nWhat are the consequences of early marriage that the authors are particularly concerned with? Is it from a public health lens, access to education, human rights, bodily autonomy, gender equality? Explicitly stating why and how child marriage is a problem in West Africa would strengthen this section.\n\nEarly Marriage in The Gambia:\n\nIt might be necessary to note that many women and adolescents of childbearing age from rural Gambia are only recently getting birth certificates, where their age is estimated from memory since the date wasn’t recorded on their actual date of birth.\n\nEspecially for women who need a birth certificate to access an antenatal card, We have observed that since now most everyone knows that child marriage is illegal, they report being 18 years of age when in actuality they are much younger. Therefore, the reported rate of 30% here is unlikely to be accurate.\n\nHigh awareness but low uptake of family planning among adolescents: A major factor seems to be people’s preference to use family planning only for birth spacing, rather than for delaying first pregnancy.\n\nMethods:\nIn the first paragraph on page 6, rephrase: “Parental informed consent was sought for minors under the age of 18 years. Since the research team was composed of men, permission was also taken from husbands of married female adolescents because culturally, husbands are expected to give permission for other men to speak with their wives.”\n\nThe qualitative component of the study is minimal. With only two focus groups, the authors are not in a position to explain differences in marriage patterns between ethnic groups. If this were a major objective of the study, in-depth interviews with different family members including elders would be necessary. Therefore, it is unclear what is gained from the focus groups. Certainly, only limited qualitative data are presented in the Results section.\n\nThe description of the qualitative data analysis is very generic.\n\nResults:\nGood break down of demographic characteristics. The rationale behind why these characteristics were selected is also strong and clear.\n\nThe finding about Wolof ethnicity having an independent effect on the probability of marrying early is interesting because they are the only ethnic group that does not typically practice female genital cutting (FGC), and the fear/ concern that girls will engage in premarital sex is among the main reasons for practicing both FGC and early marriage.\n\nThis study brings up further questions about the relationship between practicing early marriage and practicing FGC – for example, are groups that do not practice FGC more likely to practice early marriage because they are not using FGC as a tool to prevent premarital sex? This points again to the fact that conducting only two focus groups, and no interviews, provides little insight into the situation. The authors do not present qualitative data that explain the reasons for differences between the ethnic groups with any detail or clarity.\n\nDiscussion:\nIn the “Early Marriage in The Gambia” section, the writers brought up that awareness of family planning methods is high but uptake is low among adolescents. The findings may suggest that shifting socials norms surrounding how and why family planning is used (i.e. currently only for birth spacing rather than delaying first pregnancy) may also be an important programmatic implication.\n\nThe weakness and limited nature of the qualitative data collection make it difficult to arrive at robust conclusions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1949
https://f1000research.com/articles/7-1767/v1	08 Nov 18	{ "type": "Method Article", "title": "Do you cov me? Effect of coverage reduction on species identification and genome reconstruction in complex biological matrices by metagenome shotgun high-throughput sequencing", "authors": [ "Federica Cattonaro", "Alessandro Spadotto", "Slobodanka Radovic", "Fabio Marroni", "Alessandro Spadotto", "Slobodanka Radovic" ], "abstract": "Shotgun metagenomics sequencing is a powerful tool for the characterization of complex biological matrices, enabling analysis of prokaryotic and eukaryotic organisms in a single experiment, with the possibility of de novo reconstruction of the whole metagenome or a set of genes of interest. One of the main factors limiting the use of shotgun metagenomics on wide scale projects is the high cost associated with the approach. However, we demonstrate that—for some applications—it is possible to use shallow shotgun metagenomics to characterize complex biological matrices while reducing costs. Here we compared the results obtained on full size, real datasets with results obtained by randomly extracting a fixed number of reads. The main statistics that were compared are alpha diversity estimates, species abundance, and ability of reconstructing the metagenome in terms of length and completeness. Our results show that a classification of the communities present in a complex matrix can be accurately performed even using very low number of reads. With samples of 100,000 reads, the alpha diversity estimates were in most cases comparable to those obtained with the full sample, and the estimation of the abundance of all the present species was in excellent agreement with those obtained with the full sample. On the contrary, any task involving the reconstruction of the metagenome performed poorly, even with the largest simulated subsample (1M reads). The length of the reconstructed assembly was sensibly smaller than the length obtained with the full dataset, and the proportion of conserved genes that were identified in the meta-genome was drastically reduced compared to the full sample. Shallow shotgun metagenomics can be a useful tool to describe the structure of complex matrices, but it is not adequate to reconstruct de novo—even partially—the metagenome.", "keywords": [ "high-throughput sequencing", "metagenome", "metagenomics", "next generation sequencing", "alpha diversity", "complex matrices" ], "content": "Introduction\n\nShotgun metagenomics offers the possibility to assess the complete taxonomic composition of biological matrices and to estimate the relative abundances of each species in an unbiased way1,2. It allows for agnostic characterization of complex communities containing eukaryotes, fungi, bacteria and also viruses, using both DNA and RNA as a starting material. In addition, the whole metagenome approach can be used not only to simply identify DNA and RNA virus in a complex matrix, but also to study the genetic diversity in virus populations3–5, and to identify potential adventitious agents in biopharmaceutical manufacturing6,7.\n\nMetagenome shotgun high-throughput sequencing has progressively gained popularity in parallel with the advancing of next-generation sequencing technologies8,9, which provide more data in less time at a lower cost than previous sequencing techniques. This allows the extensive application to study the most various biological mixtures such as environmental samples10,11, gut samples12–14, skin samples15, clinical samples for diagnostics and surveillance purposes16,19, food ecosystems20,21 and drugs manufactured using biological sources as vaccines22.\n\nThe aim of whole metagenome approaches is not only to study the taxonomic composition of biological substrates but also to identify which genes and metabolic pathways are present with the aim to understand functional capacities in the studied microbiota13,23. Recently the approach has been also used to analyze the ensemble of genes that may encode antibiotic resistance in various microbial ecosystems (i.e. soil), which are defined as the resistome24.\n\nAnother, more traditional approach currently used to assign taxonomy to DNA sequences is based on the sequencing of target conserved regions. Metabarcoding method relies on conserved sequences to characterize communities of complex matrices. These include the highly variable region of 16S rRNA gene in bacteria27, the nuclear ribosomal internal transcribed spacer (ITS) region for fungi28, 18S rRNA gene in eukaryotes29, cytochrome c oxidase sub-unit I (COI or cox1) for taxonomical identification of animals30, rbcL, matK and ITS2 as the plant barcode31. Considering the large amount of genetic diversity within and between virus families, a universal metabarcoding approach is not applicable to detect virus nucleic acids in complex biological samples.\n\nThe selection of conserved regions has the advantage of reducing sequencing needs, since it does not require sequencing of the full genome, just a small region. On the other hand, given the currently used approaches, characterization of microbial and eukaryotic communities requires different primers and library preparations32. In addition, several studies suggested that whole shotgun metagenome sequencing is more effective in the characterization of metagenomics samples compared to target amplicon approaches, with the additional capability of providing functional information regarding the studied sample33.\n\nCurrent whole shotgun metagenome experiments are performed obtaining several million reads10,13. However, obtaining a broad characterization of the relative abundance of different species, might easily be achieved with lower number of reads.\n\nTo test this hypothesis, we performed sequencing using whole metagenomics approach of seven samples derived from different complex matrices to characterize their composition, and subsequently tested the accuracy of several measures when downsampling the number of reads used for analysis including the performance of de novo assembly in the ability to reconstruct both entire genomes and genes.\n\n\nMethods\n\nThe following samples were used in the present work: two samples collected from a live attenuated virus vaccine (B1 and B2), two horse fecal samples (F1 and F2), and three food samples (M1, M2, and M3).\n\nBiological medicines were two different lots of live attenuated MPRV vaccine (Prorix Tetra, Glaxo SmithKline) widely used for immunisation against measles, mumps, rubella and chickenpox in infants. Lyophilised vaccines were resuspended in 500 μl sterile water for injection and DNA extracted using Maxwell® 16 Instrument and the Maxwell® 16 Tissue DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions.\n\nHorse feces from two individuals were collected as follows: 100 mg of starting material stored in 70% ethanol were processed for DNA extraction using the QIAamp PowerFecal DNA Kit (QIAGEN GmbH, Hilden, Germany), according to the manufacturer's instructions.\n\nFood samples were raw materials of animal and plant origin, used to industrially prepare bouillon cubes. DNA extractions from those three samples were performed starting from 2 grams of material each, using the DNeasy mericon Food Kit (QIAGEN GmbH, Hilden, Germany), according to the manufacturer's instructions.\n\nDNA purity and concentration were estimated using a NanoDrop Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA) and Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA).\n\nDNA library preparations were performed according to manufacturer’s protocol, using the kit Ovation® Ultralow System V4 1–96 (Nugen, San Carlos, CA). Library prep monitoring and validation were performed both by Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA) and Agilent 2100 Bioanalyzer DNA High Sensitivity Analysis kit (Agilent Technologies, Santa Clara, CA).\n\nCluster generation, template hybridization, isothermal amplification, linearization, blocking and denaturization and hybridization of the sequencing primers was then performed on Illumina cBot and flowcell HiSeq SBS V4 250 cycle kit, loaded on HiSeq2500 Illumina sequencer producing 125bp paired-end reads (for samples B1, B2, M1, M2 and M3) and 250bp paired-end reads (for samples F1 and F2).\n\nThe CASAVA Illumina Pipeline version 1.8.2 was used for base-calling and de-multiplexing. Adapters were masked using cutadapt34. Masked and low quality bases were filtered using erne-filter version 1.4.635.\n\nThe bioinformatics analysis performed in the present work are summarized in Figure 1.\n\nDifferent read lengths among samples may constitute an additional confounder in analysis. To obtain homogeneous read length across samples, reads sequenced belonging to F1 and F2 were trimmed to a length of 125 bp using fastx-toolkit version 0.0.13 before subsequent analysis.\n\nReduction in coverage was simulated by randomly sampling a fixed number of reads from the full set of reads. Subsamples of 10,000, 25,000, 50,000, 100,000, 250,000, 500,000 and 1,000,000 reads were extracted from the raw reads using seqtk version 1.3. To assess variability, subsampling was performed 5 times for each sample and forr each read abundance.\n\nClassification of reads against the NCBI nt database downloaded on May 2018 was performed using Kraken 2 version 2.0.6-beta36 to estimate species abundance and Shannon diversity index. A simplified representation of species composition was obtained using Krona version 2.637.\n\nChao138 species richness and Shannon’s diversity39 were estimated using the R package vegan version 2.4.240.\n\nAssembly of the metagenome was performed using megahit version 1.1.241. Completeness of the assembly was assessed using BUSCO version 3.0.242. The proportion of the reconstructed genes was measured as the proportion of genes that were fully reconstructed, plus the proportion of genes that were partially reconstructed. BUSCO analysis was performed on prokaryotic database for all the samples with the exception of M1 (mostly composed by fungi) for which the fungal database was used. Samples B1 and B2 were also compared against the eukaryotic BUSCO database; results for the prokaryotic database are reported.\n\nUnless otherwise specified, all the analysis were performed using R 3.3.343.\n\n\nResults\n\nSummary statistics for the full samples included in the study are shown in Table 1.\n\nN species, number of species identified in the sample including species identified by one or more reads; Singletons, number of species identified in the sample by only one read.\n\nThe number of reads obtained in the samples selected for the present study ranged from slightly more than 1 million (sample M2) to more than 12 million (sample F1). Our subsampling, ranging from 10,000 to 1,000,000 reads, led to a reduction in size of 36% (1,000,000 out of 1,558,975) in M2 to 0.08% of the original size (10,000 reads out of 12,472,553) in F1.\n\nSamples used in this study had different levels of species composition (Figure 2). Some samples, such as M1, B1 and B2 were dominated by a single species, while others, in particular fecal samples, showed high heterogeneity in species composition.\n\nFigure 3 shows the variation of the value of Chao1 estimator, representing the estimated number of species in each sample when varying the number of reads used for the estimation, from the smallest number on the left, to the full dataset on the right. The value of Chao1 estimator for the full dataset is plotted on the right side of the plot, at the rightmost fecal samples F1 and F2 had an estimated number of species greater than 40,000, much higher than all the other samples, for which less than 20,000 species were estimated (less than 10,000 B1, B2, M1 and M3).\n\nX axis is in log scale, Y axis is in linear scale. Shaded areas represent the confidence limits of resampling experiments. “Full” represents the values obtained with the full set of reads (number of reads per sample listed in column 2 of Table 1).\n\nThe effect of downsampling on the estimated number of species has different effects in different samples. For most samples, even a robust downsampling led to only a slight reduction in the estimated species richness. However, for samples F1 and F2, which were characterized by a high number of overall species and rare species, the downsampling led to a significant reduction in the estimated species richness.\n\nShannon’s diversity index is a widely used method to assess the biological diversity of ecological and microbiological communities. Figure 4 depicts the effect of subsampling on the Shannon’s diversity index. The effect of subsampling on Shannon’s diversity index is smaller than the effect on the estimated number of species. The variation in Shannon diversity index with subsampling is negligible for all samples, even reducing the number of reads from the full size to 100,000 or less.\n\nX axis is in log scale, Y axis is in linear scale. Shaded areas represent the confidence limits of resampling experiments. “Full” represents the values obtained with the full set of reads (number of reads per sample listed in column 2 of Table 1).\n\nFigure 5 shows the correlation in species abundance estimation between the full dataset and a reduced dataset of 100,000 reads. The linear correlation coefficient between the two datasets is >0.99 in all the replicates. The plot is in log-log scale to emphasize differences in low abundance species. Only species with frequencies lower than 0.01% (i.e. species represented in 1 read out of 10,000) show some effect of subsampling on the relative abundance estimation. All the seven samples share a similar behavior.\n\nData for all the five replicates of the subsampling are plotted. Each point (colored by sample of origin) represents a given species. The position on the X axis represents the relative abundance of the species in the full dataset, and the position on the Y axis represents the relative abundance of the species in the samples obtained by randomly sampling 100,000 reads. Both axis are plotted in log scale to facilitate visualization of low abundance species.\n\nIn Figure 6 we show the results obtained by reducing the number of sampled reads to 10,000 reads per sample. Similar to what we observed for larger subsamples, the linear correlation coefficient between species abundance estimate in the full and the reduced dataset was high in all the samples (r>0.95) and in all the replicated subsampling. The abundance of species with frequency greater than 1/1000 (0.1%) is correctly estimated in the subsamples, while for rare species the estimate is not precise. Species with frequencies <0.01% are by definition absent in the subsample obtained with 10,000 reads, and were arbitrarily set to a frequency of 0.001% to provide the reader with an idea on their abundance and distribution in the original sample.\n\nData for all the five replicates of the subsampling are plotted. Each point (colored by sample of origin) represents a given species. The position on the X axis represents the relative abundance of the species in the full dataset, and the position on the Y axis represents the relative abundance of the species in the samples obtained by randomly sampling 10,000 reads. Both axis are plotted in log scale to facilitate visualization of low abundance species.\n\nWhile characterizing and measuring species present in a complex matrix is an important task, some studies aim at reconstructing (partially or entirely) the metagenome via a de novo approach. We thus investigated the effect of coverage reduction on this task. We reconstructed de novo the metagenome of the full and reduced datasets, and compared the reconstructed genomes. Results are summarized in Figure 7. As expected, the size of the assembly is strongly influenced by the read number. Assemblies obtained using the full set of reads had a size ranging from slightly more than 1 Mb (sample B1) to nearly 100 Mb (F1 and F2). A decrease in the number of reads used for the assembly lead to a steady decrease in assembly size in all samples, although with different slopes. Assembly sizes obtained using 1,000,000 reads ranged from less than 1 Mb (F1 and F2) to slightly more than 10 Mb (M1), and those obtained using 100,000 reads ranged from less than 100 Kb (F1 and F2) to less than 1 Mb (all the remaining samples).\n\nX and Y axes are in log scale. Shaded areas represent the confidence limits of resampling experiments. “Full” represents the values obtained with the full set of reads (number of reads per sample listed in column 2 of Table 1).\n\nHowever, the total assembly length is not necessarily a sufficient measure to describe assembly goodness and completeness42,44. Since we are interested in assessing the completeness of the reconstructed metagenome, we used BUSCO to report the proportion of genes covered by any given assembly42. Figure 8 reports the proportion of metagenome completeness estimated by BUSCO from full and from the reduced dataset obtained by randomly sampling 1,000,000 reads. The prokaryotic BUSCO dataset was used for all samples with the exception of sample M1, composed prevalently by a mushroom, for which the fungal BUSCO database was used. The full samples F1 and F2 reconstructed a fairly complete proportion of the BUSCO genes (>90%), while the reduced dataset reconstructed less than 20%.\n\nError bars are based on the five replicate experiments performed for each sample.\n\nSimilar trends can be observed with other datasets. Given the lower number of reads sequenced in other samples, the performance in reconstructing the BUSCO genes was generally poor, but reducing to 1 million reads led to a further decrease in performance, suggesting that this is a clearly suboptimal number of reads. Samples B1 and B2 show a very poor performance because the prokaryotic organisms in the sample are very rare contaminants. Being derived from fetal human cell cultures, a large portion of the metagenome is constituted by human sequences, but given the very small ability in reconstructing de novo a genome as large as the human one, the proportion of reconstructed BUSCO genes is very low (<5% both for prokaryotic and eukaryotic BUSCO genes).\n\n\nDiscussion\n\nThe aim of the present work was to assess the reliability of low-depth shotgun metagenome sequencing for the characterization of complex matrices, as follows: 1) determining diversity and species richness in complex matrices; 2) estimating abundance of the species present in the complex matrix, and 3) reconstructing de novo the genome of the species present in the samples. We selected seven heterogeneous complex samples, sequenced at varying coverage (ranging 1 to 12 million reads). Shotgun metagenomics experiments—often aiming at reconstructing de novo the studied metagenome—have a tendency to generate a very high number of reads per sample10. Compared to such studies, all of our samples have relatively shallow coverage of the metagenome, and we tested if even lower coverage could still provide reliable answers to the three main questions listed above.\n\nWe used Chao1 as an indicator of species richness and Shannon’s diversity index as an estimator of species diversity, and we measured their variation when reducing the number of reads used for the experiment.\n\nAn important detail to be considered here is the fact that the two indices behave differently in the full and the reduced samples. We provide an explanation regarding the reasons of this difference.\n\nChao1 estimator is obtained as\n\n\n\nWhere Sobs is the number of observed species in the sample, f1 is the number of species observed once, and f2 is the number of species observed twice.\n\nShannon diversity index is estimated as\n\n\n\nWhere N is the total number of species and pi is the frequency of the species i.\n\nThus, the Chao1 index is heavily affected by the number of rare species that are identified and not from the relative frequencies of the most abundant species, while the Shannon diversity index is affected more by variation in the frequencies of highly abundant species than by the disappearance of rare species.\n\nSamples F1 and F2 are characterized by a very large number of observed species (29,661 and 25,608, respectively), while all the other samples have lower number of species, ranging from 2508 in B1 to 9638 in M2. Chao1 captures this differences, showing that F1 and F2 have greater diversity estimates. The Shannon diversity index, on the contrary, relies not only on the number of observed species, but also on the frequency distribution, and for a given number of species reaches its maximum for equifrequent species. Therefore, samples that have a relatively high number of common species with comparable frequencies tend to have high Shannon’s diversity indices.\n\nAs an example the number of species with a frequency greater than 0.1% was 23 in sample F1 and was 55 in sample M2. Thus, in spite of a much lower number of species in M2 compared to F1, the Shannon diversity is higher in M2 than in F1. Given the differences in behavior between the two indices in certain conditions, we decided to use both of them to have a more complete information on sample diversity when decreasing coverage. Our results show that a substantial reduction of coverage can be safely achieved without compromising the ability of estimating species richness and abundance (Figure 3 and Figure 4), although the estimated number of species is moderately affected by coverage reduction.\n\nWe then set out to assess the changes in the estimated relative frequency of each individual species when reducing the number of sequenced reads. Accurate estimate of the relative abundance of each species is an important task when the aim is a) to detect species with a relative abundance above any given threshold, b) to differentiate two samples based on different abundance of any given species composition, or c) to cluster samples based on their species composition. Our results show that even reducing sequencing to 100,000 reads, species abundances as low as 0.01% can be reliably estimated.\n\nThe last questions to which we sought to answer is if a reduction in the sequencing coverage would have a deleterious effect on the ability of de novo assembling the metagenome. Our results show that downsampling had a strongly negative effect on the total length of the reconstructed metagenome and on the proportion of BUSCO genes reconstructed with the metagenome assembly.\n\nBUSCO is widely used for assessing the completeness of genome and transcriptome assemblies for individual organisms, and has benchmark datasets for several lineages. It is possible that using BUSCO for assessing completeness of a metagenomics assembly, including both eukaryotic and prokaryotic organisms, results in an underestimation of the completeness of the reconstruction. However, the aim of the present work is not the absolute estimation of the completeness of the metagenomics assembly, but rather the relative variation observed when using a subsample of reads. Our results indicate that even using 1,000,000 reads is clearly suboptimal in terms of fully sampling the genes present in the complex matrices. This observation needs to be taken into account in the phase of experimental design. Our conclusions also affect research aimed at reconstruction of an interesting part of the meta-genome, such as genes involved in antibiotic resistance24. The decrease in performance observed in the reconstruction of BUSCO genes will be likely observed for the reconstruction of other gene categories. Researchers aiming at a de novo reconstruction of the metagenome (although partial) must keep in mind that several millions of reads are needed to attain reliable results.\n\nIn the present work we tested the feasibility of using metagenome shotgun shallow high-throughput sequencing to analyze complex samples for the presence of eukaryotes, prokaryotes and virus nucleic acids with the aim of monitoring, diagnosis, surveillance, quality control and traceability.\n\nWe show that, if the aim of the experiment is a taxonomical characterization of the sample or the identification and quantification of species present in it, then a low-coverage WGS is a good choice. On the other hand, if one of the aims of the study relies on de novo assembly, then a higher number of reads is required. We do not provide here a suggestion on the number of reads that are needed when the aim is the (partial) reconstruction of the meta-genome, as it depends on several factors (number of species in the sample, their genome size, and their abundance, length of the sequencing reads, quality of the DNA) and this estimation needs to be performed for each experiment based on detailed understanding of the experiment aims and of sample characteristics.\n\n\nData availability\n\nRaw reads are available at NCBI Sequence Read Archive. Samples F1 and F2 are available under accession number SRP163102: https://identifiers.org/insdc.sra/SRP163102; samples B1 and B2 are available under accession number SRP163096: https://identifiers.org/insdc.sra/SRP163096; and samples M1, M2 and M3 are available under accession number SRP163007: https://identifiers.org/insdc.sra/SRP163007.", "appendix": "Grant information\n\nMetagenome sequencing of B1 and B2 (MPRV vaccines, Prorix Tetra, GlaxoSmithKline) was financed by Corvelva (non-profit association, Veneto, Italy), in the frame of a contract work with IGA Technology Services. No other grants were involved in supporting the work.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe authors would like to thank Dr Loretta Bolgan for fruitful scientific discussions and Corvelva (non-profit association, Veneto, Italy) to give us the permission to use their own metagenome sequencing data (samples B1 and B2) for the paper purposes; Dr Federica Cattapan (Mérieux NutriSciences Italia and Chelab S.r.l., Italia) to provide the DNAs of M1, M2, M3 samples and Dr Carol Hughes (Phytorigins Ltd., United Kindom) to give us the biological samples F1, F2 and to both of them to give us the permission to use their samples for whole metagenome sequencing and analysis.\n\n\nReferences\n\nQuince C, Walker AW, Simpson JT, et al.: Shotgun metagenomics, from sampling to analysis. Nat Biotechnol. 2017; 35(9): 833–44. PubMed Abstract \| Publisher Full Text\n\nForbes JD, Knox NC, Ronholm J, et al.: Metagenomics: The Next Culture-Independent Game Changer. Front Microbiol. 2017; 8: 1069. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEdwards RA, Rohwer F: Viral metagenomics. Nat Rev Microbiol. 2005; 3(6): 504–10. PubMed Abstract \| Publisher Full Text\n\nSahoo MK, Holubar M, Huang C, et al.: Detection of Emerging Vaccine-Related Polioviruses by Deep Sequencing. McAdam AJ, editor. J Clin Microbiol. 2017; 55(7): 2162–71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMartí JM: Robust Analysis of Time Series in Virome Metagenomics. Methods Mol Biol. 2018; 1838: 245–60. PubMed Abstract \| Publisher Full Text\n\nRichards B, Cao S, Plavsic M, et al.: Detection of adventitious agents using next-generation sequencing. PDA J Pharm Sci Technol. 2014; 68(6): 651–60. PubMed Abstract \| Publisher Full Text\n\nPetricciani J, Sheets R, Griffiths E, et al.: Adventitious agents in viral vaccines: lessons learned from 4 case studies. Biologicals. 2014; 42(5): 223–36. PubMed Abstract \| Publisher Full Text\n\nBragg L, Tyson GW: Metagenomics using next-generation sequencing. Methods Mol Biol. 2014; 1096: 183–201. PubMed Abstract \| Publisher Full Text\n\nDesai N, Antonopoulos D, Gilbert JA, et al.: From genomics to metagenomics. Curr Opin Biotechnol. 2012; 23(1): 72–6. PubMed Abstract \| Publisher Full Text\n\nSunagawa S, Coelho LP, Chaffron S, et al.: Ocean plankton. Structure and function of the global ocean microbiome. Science. American Association for the Advancement of Science; 2015; 348(6237): 1261359. PubMed Abstract \| Publisher Full Text\n\nWilhelm RC, Cardenas E, Leung H, et al.: A metagenomic survey of forest soil microbial communities more than a decade after timber harvesting. Sci data. Nature Publishing Group; 2017; 4: 170092. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHamady M, Knight R: Microbial community profiling for human microbiome projects: Tools, techniques, and challenges. Genome Res. 2009; 19(7): 1141–52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQin J, Li R, Raes J, et al.: A human gut microbial gene catalogue established by metagenomic sequencing. Nature. Nature Publishing Group; 2010; 464(7285): 59–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuman Microbiome Project Consortium: Structure, function and diversity of the healthy human microbiome. Nature. Nature Publishing Group; 2012; 486(7402): 207–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOh J, Byrd AL, Deming C, et al.: Biogeography and individuality shape function in the human skin metagenome. Nature. Nature Publishing Group; 2014; 514(7520): 59–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilson MR, Suan D, Duggins A, et al.: A novel cause of chronic viral meningoencephalitis: Cache Valley virus. Ann Neurol. 2017; 82(1): 105–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilson MR, Naccache SN, Samayoa E, et al.: Actionable diagnosis of neuroleptospirosis by next-generation sequencing. N Engl J Med. Massachusetts Medical Society; 2014; 370(25): 2408–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGreninger AL, Messacar K, Dunnebacke T, et al.: Clinical metagenomic identification of Balamuthia mandrillaris encephalitis and assembly of the draft genome: the continuing case for reference genome sequencing. Genome Med. 2015; 7(1): 113. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nForbes JD, Knox NC, Peterson CL, et al.: Highlighting Clinical Metagenomics for Enhanced Diagnostic Decision-making: A Step Towards Wider Implementation. Comput Struct Biotechnol J. Elsevier; 2018; 16: 108–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMayo B, Rachid CT, Alegria A, et al.: Impact of next generation sequencing techniques in food microbiology. Curr Genomics. 2014; 15(4): 293–309. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOniciuc EA, Likotrafiti E, Alvarez-Molina A, et al.: The Present and Future of Whole Genome Sequencing (WGS) and Whole Metagenome Sequencing (WMS) for Surveillance of Antimicrobial Resistant Microorganisms and Antimicrobial Resistance Genes across the Food Chain. Genes (Basel). 2018; 9(5): pii: E268. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVictoria JG, Wang C, Jones MS, et al.: Viral nucleic acids in live-attenuated vaccines: detection of minority variants and an adventitious virus. J Virol. 2010; 84(12): 6033–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDenman SE, Morgavi DP, McSweeney CS: Review: The application of omics to rumen microbiota function. Animal. 2018; 1–13. PubMed Abstract \| Publisher Full Text\n\nAdu-Oppong B, Gasparrini AJ, Dantas G: Genomic and functional techniques to mine the microbiome for novel antimicrobials and antimicrobial resistance genes. Ann N Y Acad Sci. 2017; 1388(1): 42–58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStaats M, Arulandhu AJ, Gravendeel B, et al.: Advances in DNA metabarcoding for food and wildlife forensic species identification. Anal Bioanal Chem. Springer Berlin Heidelberg; 2016; 408(17): 4615–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYamamoto S, Masuda R, Sato Y, et al.: Environmental DNA metabarcoding reveals local fish communities in a species-rich coastal sea. Sci Rep. Nature Publishing Group; 2017; 7(1): 40368. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCaporaso JG, Lauber CL, Walters WA, et al.: Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci U S A. 2011; 108 Suppl 1: 4516–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchoch CL, Seifert KA, Huhndorf S, et al.: Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci U S A. National Academy of Sciences; 2012; 109(16): 6241–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHugerth LW, Muller EE, Hu YO, et al.: Systematic design of 18S rRNA gene primers for determining eukaryotic diversity in microbial consortia. Voolstra CR, editor. PLoS One. Public Library of Science; 2014; 9(4): e95567. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHebert PD, Cywinska A, Ball SL, et al.: Biological identifications through DNA barcodes. Proc Biol Sci. 2003; 270(1512): 313–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFazekas AJ, Kuzmina ML, Newmaster SG, et al.: DNA barcoding methods for land plants. Methods Mol Biol. 2012; 858: 223–52. PubMed Abstract \| Publisher Full Text\n\nUyaguari-Diaz MI, Chan M, Chaban BL, et al.: A comprehensive method for amplicon-based and metagenomic characterization of viruses, bacteria, and eukaryotes in freshwater samples. Microbiome. BioMed Central; 2016; 4(1): 20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRanjan R, Rani A, Metwally A, et al.: Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing. Biochem Biophys Res Commun. NIH Public Access; 2016; 469(4): 967–77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMartin M: Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011; 17(1): 10–2. Publisher Full Text\n\nDel Fabbro C, Scalabrin S, Morgante M, et al.: An extensive evaluation of read trimming effects on Illumina NGS data analysis. Seo JS, editor. PLoS One. Public Library of Science; 2013; 8(12): e85024. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWood DE, Salzberg SL: Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol. BioMed Central; 2014; 15(3): R46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOndov BD, Bergman NH, Phillippy AM: Interactive metagenomic visualization in a Web browser. BMC Bioinformatics. 2011; 12(1): 385. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChao A: Non-parametric estimation of the classes in a population. Scand J Statist. Scandinavian Journal of Statistics; 1984; 11(4): 265–70. Reference Source\n\nShannon CE: A Mathematical Theory of Communication. Bell Syst Tech J. 1948; 27(3): 379–423. Publisher Full Text\n\nOksanen J, Blanchet G, Friendly M, et al.: vegan: Community Ecology Package. 2017. Reference Source\n\nLi D, Liu CM, Luo R, et al.: MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics. 2015; 31(10): 1674–6. PubMed Abstract \| Publisher Full Text\n\nSimão FA, Waterhouse RM, Ioannidis P, et al.: BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. Oxford University Press; 2015; 31(19): 3210–2. PubMed Abstract \| Publisher Full Text\n\nR Core Team: R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 2018.\n\nVezzi F, Narzisi G, Mishra B: Feature-by-feature--evaluating de novo sequence assembly. Rzhetsky A, editor. PLoS One. Public Library of Science; 2012; 7(2): e31002. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "40445", "date": "27 Nov 2018", "name": "Alejandro Sanchez-Flores", "expertise": [ "Reviewer Expertise Genomics", "Transcriptomics", "Metagenomics", "Bioinformatics" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors propose and evaluate a whole metagenome shotgun analysis via a low sequencing yield approach, using the Illumina platform.\nIn general, the idea and hypothesis are good, but the experimental design itself lacks important controls and there are many variables that are not analyzed and that can potentially bias the results.\nMy main concern is that the used samples have many variables and despite using a \"replicate\" for each case, samples within the same type were very different. Also the nature of each sample could have an effect in the DNA isolation, in particular for the vaccine ones. Also, regarding the vaccines, it is not clear to me, if what they are looking for is DNA of potential contaminants, since all viruses in the vaccine are ssRNA. That would be my guess, but is not clear from the text.\nThe main problem is that to test the influence of the sequencing yield, it would be extremely important to know the initial DNA concentration of each organism in the sample. Therefore, a mock metagenome or controlled sample would be much better as a reference to compare real life cases. In real life cases, the presence of certain organisms detected by the presence of its DNA, is not necessarily an indicator of the availability of alive organisms. Depending on the case, the presence of just the organism DNA could be an indicator of contamination which in the case of vaccines could be really bad. However, in the case of food material, finding DNA of pathogens, has to be associated with microbiology tests. However, with low sequencing yield, is very probable that very DNA in low amounts will be missed, even if this is not changing diversity indexes such as Chao1 and Shannon.\nFinally, the main difference where low yield has a significant impact can be observed in the fecal samples. This is expected since among all the tested samples, fecal ones are the most diverse and sub-sampling will really affect them as observed in Figure 3.\nSince the composition of each sample is not known a priori, then there are some factors that can contribute to biases. As mentioned, the DNA concentration but also its integrity (fragmentation) will affect the library construction; the cited kit requires DNA amplification which will have a bias towards GC rich genomic regions; library size was not described and was not mentioned if the samples were pooled with other libraries with different insert sizes, which affect not only the sequencing quality but the yield.\nIn terms of bioinformatics analysis, it will be required to put the parameters used for each program, in case someone wants to reproduce this. For Kraken2, it is important to know what is the kmer size to index the database. For MEGAHIT assembly it will be important to know the kmer and step sizes used. For the completeness assessment, the authors used BUSCO, but apparently they are using the whole assembly to assess the completeness. This is not correct, since they must first separate in bins which genomes they have really reconstructed and then they can assess the completeness of them. Probably they can report the an average completeness value for all the reconstructed genomes. By doing the binning they can have a better analysis of what was really reconstructed and how complete it was.\nThe use of Krona in Figure 2 is not very convenient. The whole point of a Krona graph is that is interactive. If authors want to provide the Krona data to be downloaded it would be possible and recommended. Having said that, I recommend to use bar plots to represent the relative abundance and composition of the samples at a given taxa level.\nAgain, the idea is very good but the work needs to be improved before indexing.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? No\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? No", "responses": [ { "c_id": "4267", "date": "30 Nov 2018", "name": "Federica Cattonaro", "role": "Author Response", "response": "We are grateful for the constructive comments. We agree with all of them and we are planning corrective actions, listed below.My main concern is that the used samples have many variables and despite using a \"replicate\" for each case, samples within the same type were very different. The observation is correct. Actually, the diversity of the samples was sought by purpose in order to be able to generalize the conclusions of our paper. The fact that diversity estimate and species abundance estimation remain reliable even with strong down-sampling for all of the samples is encouraging us to think that this is a general (although not necessarily universal) observation. The same is true for the observation that de-novo assembly quickly loses accuracy when decreasing the number of sequenced reads. Maybe this wasn’t made clear enough in the paper, and we will clarify it.Also the nature of each sample could have an effect in the DNA isolation, in particular for the vaccine ones. Quantities of DNA isolated from vaccine samples (B1 and B2) were estimated to be ~2 µg using Qbit fluorimeter. However, we will provide a table with all the details about quantity, concentration, quality and size of starting DNA for all samples used in the study.Also, regarding the vaccines, it is not clear to me, if what they are looking for is DNA of potential contaminants, since all viruses in the vaccine are ssRNA. That would be my guess, but is not clear from the text.The vaccine composition declared by the producer is the following:Live attenuated viruses: Measles (ssRNA) Swartz strain, cultured in embryo chicken cell cultures; Mumps (ssRNA) strain RIT 4385, derived from the Jeryl Linn strain, cultured in embryo chicken cell cultures; Rubella (ssRNA) Wistar RA 27/3 strain, grown in human diploid cells (MRC-5); Varicella (dsDNA) OKA strain grown in human diploid cells (MRC-5).By DNA-seq we expected to find Varicella (dsDNA) OKA strain DNA (which was found and confirmed by variant analysis with respect to AB097932.1 Human herpesvirus 3 DNA, sub strain vOka). In addition, we found also human and chicken DNA. For human’s, we confirmed MRC-5 cell origin by mitochondrial genome variant analysis.Genotyping analyses gave us confidence on the validity of the obtained results, even though they were beyond the scope of this work.To identify vaccine’s ssRNA viruses we extracted RNA and performed RNA-seq from the same B1 and B2 samples. This aspect also goes beyond the scope of this work.The main problem is that to test the influence of the sequencing yield, it would be extremely important to know the initial DNA concentration of each organism in the sample. Therefore, a mock metagenome or controlled sample would be much better as a reference to compare real life cases. A mock community experiment is already on-going by using ‘10 Strain Staggered Mix Genomic Material (ATCC® MSA-1001™)’. Of course, the data obtained will be integrated in the analysis results.In real life cases, the presence of certain organisms detected by the presence of its DNA, is not necessarily an indicator of the availability of alive organisms. Depending on the case, the presence of just the organism DNA could be an indicator of contamination which in the case of vaccines could be really bad. However, in the case of food material, finding DNA of pathogens, has to be associated with microbiology tests. We agree with the observation of the reviewer. However, the aim of this work is to determine if low-pass whole genome sequencing can be an appropriate approach to broadly describe a complex matrix; finding and confirming contaminants in vaccines or DNA pathogens in food samples was beyond of the scope of the paper. However, with low sequencing yield, is very probable that very DNA in low amounts will be missed, even if this is not changing diversity indexes such as Chao1 and Shannon. Finally, the main difference where low yield has a significant impact can be observed in the fecal samples. This is expected since among all the tested samples, fecal ones are the most diverse and sub-sampling will really affect them as observed in Figure 3.We agree with the reviewer; we add some thoughts just to clarify. We indeed observed that extremely rare species (with frequencies lower than 1/10000) are lost when subsampling to the most extreme levels. When subsampling to 100K reads we are losing species with a frequency around 1/100,000 (very approximate estimate). However, the effect of losing such species on the global sample diversity as estimated by Shannon diversity index is negligible (see Figure 4, in which we show that reduction in sequencing depth has no dramatic effect on Shannon’s diversity index). The situation is different for the Chao 1 estimator. This is expected and is due to the way Chao1 is computed: this estimator relies heavily on the number of singletons (i.e. species represented by only one read). By subsampling, singletons (i.e. the rarest species) are very likely to be lost. The same phenomenon can be inferred by looking at Figures 5 and 6. Those represent a scatterplot of the relative abundance of species in full sample and reduce samples (100K and 10k reads, respectively). The plots are shown in log log scale to emphasize differences for low-frequency species. Only low-frequency species have some variation in frequency estimation. However, even when sampling only 10K read, species with frequency around 0.1% (i.e. 1/1000) are appropriately quantified. All of these observations led us to conclude that coverage reduction doesn’t prevent a satisfactory characterization of complex matrices (with the only exception of Chao 1 estimator).Since the composition of each sample is not known a priori, then there are some factors that can contribute to biases. As mentioned, the DNA concentration but also its integrity (fragmentation) will affect the library construction; the cited kit requires DNA amplification which will have a bias towards GC rich genomic regions; library size was not described. The Nugen Ovation® Ultralow System V4 kit used is a standard kit for NGS library preparation (https://www.nugen.com/sites/default/files/DS_v2-Ovation_Ultralow_V2.pdfIt is a standard protocol widely used by the scientific community to perform DNA-seq also from low input DNA quantities (1 ng), even if in our case input DNA was of moderate quantity. Mock community experiment will shed light on eventual biases.DNA concentration and integrity as well as input DNA quantities used in library construction and libraries insert size will be reported in the version 2 of the paper.It was not mentioned if the samples were pooled with other libraries with different insert sizes, which affect not only the sequencing quality but the yield.Samples were sequenced in different runs and pooled with other libraries of similar insert sizes. The number of reads obtained per sample reflects and respects their quantities, i.e. nmols that were loaded on the sequencer.In terms of bioinformatics analysis, it will be required to put the parameters used for each program, in case someone wants to reproduce this. For Kraken2, it is important to know what is the kmer size to index the database. For MEGAHIT assembly it will be important to know the kmer and step sizes used. All these details will be provided in the version 2 of the paper.For the completeness assessment, the authors used BUSCO, but apparently they are using the whole assembly to assess the completeness. This is not correct, since they must first separate in bins which genomes they have really reconstructed and then they can assess the completeness of them. Probably they can report the an average completeness value for all the reconstructed genomes. By doing the binning they can have a better analysis of what was really reconstructed and how complete it was.This is a good point. While our aim was to estimate the total proportion of BUSCO genes that were reconstructed, irrespective of the species of the organism to which they belong, we understand that a practical application is likely to require separating the reconstructed genomes. We will integrate our analysis by binning the reconstructed genomes.The use of Krona in Figure 2 is not very convenient. The whole point of a Krona graph is that is interactive. If authors want to provide the Krona data to be downloaded it would be possible and recommended. Having said that, I recommend to use bar plots to represent the relative abundance and composition of the samples at a given taxa level.We will either provide a link to interactive krona graphs and/or bar plots reporting the relative abundance and composition of the samples." } ] }, { "id": "42422", "date": "04 Jan 2019", "name": "José F. Cobo Diaz", "expertise": [ "Reviewer Expertise microbial ecology", "metabarcoding sequencing", "NGS data analysis", "bacterial communities", "fungal communities" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors proposed and evaluated the influence of reduce sequencing effort (amount of sequences) for a whole metagenome shotgun analysis, using the Illumina platform, in the species composition and diversity index of the communities studied. Although the idea and hypothesis are good, some problems were found in the experimental design and data analysis.\n\nAccording to the questions proposed in the peer review form, it is not a new method, only the adaptation of a current methodology to optimize the cost and increase the potential numbers of samples analyzed per run of Illumina platform. Although the introduction is clearly explained, the reasons for use shotgun sequencing, mainly to analyze viruses data and functional data for all the organism, no emphasis on such points was done in the results and discussion. The samples used (vaccines, horse fecal samples and food samples) and the introduction remark the detection of pathogens as the main objective of the approach used, including viruses, which can not be screened by amplicons approaches, like metabarcoding sequencing. I suggest adapting the text and manuscript to focus on pathogens (mainly viruses) found along the sub-samples taken for each sample. At that point, some contaminated samples (or not contaminated samples mixed with known amounts DNA from pathogen viruses) have to be used to determine the lowest pathogen concentration that could be detected for each shotgun sequencing coverage proposed.\n\nMany problems were found with the methodology employed, mainly the parameters used in each step and/or software employed for data filtering and analysis, which are critical for the results, which can have strong variations depending of the parameters used. Hence, the methodology proposed does not allow any replication of the method used. Moreover, there are some mistakes for species designation in the study, with at least 2508 species found in vaccine samples indicating big problems along read filtering and data analysis, because this number of species is often found in more complex systems, such as soils samples from agricultural fields. Moreover, go to species classification using some taxonomical markers, such ITS or 16SrRNA, is risky with sequences lower than 400 bp, and sometimes with bigger sequences. In the current manuscript, the use of non taxonomical marker sequences and 150 bp lengths increase enormously the number of sequences not correctly assigned to species level, and in several cases also for higher taxonomical levels (genus, family...). Therefore, I suggest to clarify how the species assignment was done, because it looks like that each gene-species was considered as one species, and each gene found for a single species was counted as a new species.\n\nAlpha diversity indexes employed are not the best ones, in my opinion, to describe or compare the sub-samples proposed in this manuscript. The chao1 index, an estimator of richness, has a strong influence on the number of singletons obtained in the samples, which due to the complexity of the samples-data tends to be high. Shannon index is influenced by both richness (number of taxa) and evenness (equability, Pielou index), and the reduction of richness due to the loss of rare taxa has a strong influence on this index. I propose to use the number of observed taxa instead of estimated taxa, and any evenness index, like the Pielou index, instead of the Shannon index. Moreover, the use of a coverage index, such Good’s coverage index, could be useful to compare the loss of information associated to sampled size or coverage.\nIn conclusion, although the raw data can contains some important information, the manuscript has to be improved with new “pathogen contaminated” samples, and be re-written to focus on the detection of pathogens in the samples, which due to the low abundance of the samples could not be detected depending of the shotgun coverage.\n\nIs the rationale for developing the new method (or application) clearly explained? No\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? No\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/7-1767
https://f1000research.com/articles/9-33/v1	22 Jan 20	{ "type": "Method Article", "title": "Using the “Uniform Scale” to facilitate meta-analysis where exposure variables are qualitative and vary between studies – methodology, examples and software", "authors": [ "Peter N Lee", "Jan Hamling", "John S Fry", "Sonja Vandyke", "Rolf Weitkunat", "Jan Hamling", "John S Fry", "Sonja Vandyke", "Rolf Weitkunat" ], "abstract": "Meta-analyses often combine covariate-adjusted effect estimates (odds ratios or relative risks) and confidence intervals relating a specified endpoint to a given exposure. Standard techniques are available to do this where the exposure is a simple presence/absence variable, or can be expressed in defined units. However, where the definition of exposure is qualitative and may vary between studies, meta-analysis is less straightforward. We introduce a new “Uniform Scale” approach allowing expression of effect estimates in a consistent manner, comparing individuals with the most and least possible exposure.\n\nIn 2008, we presented methodology and made available software to obtain estimates for specific pairwise comparisons of exposure, such as any versus none, where the source paper provides estimates for multiple exposure categories, expressed relative to a common reference group. This methodology takes account of the correlation between the effect estimates for the different levels. We have now extended our software, available in Excel, SAS and R, to obtain effect estimates per unit of exposure, whether the exposure is defined or is to be expressed in the “Uniform Scale”. Examples of its use are presented.", "keywords": [ "systematic review", "meta-analysis", "contrast", "dose response" ], "content": "Introduction\n\nResults from individual studies relating an exposure of interest to risk of a disease are often recorded as a set of covariate-adjusted effect estimates (odds ratios (ORs), or relative risks (RRs)), with 95% confidence intervals (CIs), for differing levels of exposure relative to a reference (base) level. Associated with this, data on the numbers of subjects are often presented. For case-control or cross-sectional studies these are subdivided by presence of disease. For prospective studies the numbers with disease and the numbers at risk are typically presented.\n\nFor the purpose of conducting meta-analyses, it is often the situation that meta-analysts with no access to the raw data of a study require estimates of covariate-adjusted ORs or RRs for pairwise comparisons other than those presented. For example, if the base level (0) is never smokers, and levels 1 to 4 are, respectively, former smokers and current smokers of 1–10, 11–20 and 21+ cigarettes per day, one may wish to derive estimates for current vs never (levels 2 to 4 combined vs level 0), current vs non (levels 2 to 4 combined vs levels 0 and 1 combined) or current vs former (levels 2 to 4 combined vs level 1). As the effect estimates (OR or RR) for each level are not independent, having a common reference level, one cannot derive these estimates straightforwardly. Thus, for example, using fixed-effects meta-analysis to combine effect estimates for levels 2 to 4 to get an estimate for current smoking is not correct.\n\nA solution to this problem, described in a paper we wrote in 2008 (Hamling et al., 2008), is based on methodology developed much earlier by Greenland & Longnecker (1992). Where there are k exposure levels, the method involves deriving, using the effect estimates and their 95% CIs together with the marginal totals of numbers of subjects, a set of pseudo-numbers for the relevant 2 x (k + 1) table. These pseudo-numbers (which have no direct meaning by themselves) produce the same effect estimates and 95% CIs for comparison with the reference level, and can be combined as appropriate to produce an adjusted estimate for any pairwise comparison of different sets of levels. Our earlier paper (Hamling et al., 2008) gives examples of the methodology in action. That paper not only shows how relative effect estimates can be derived for alternative comparisons, but also presents methodology for deriving alternative comparisons when results are given by categories of disease rather than categories of exposure. It also makes available software to derive the necessary estimates using both an Excel and a SAS implementation. These implementations also produce chi-squared and p-values for heterogeneity and trend corresponding to the table of pseudo-numbers of subjects and based on trend coefficients entered by the user, using formulae 4.38 and 4.39 of Breslow & Day (1980) for case-control studies and modified versions of these formulae for prospective studies described in their later publication (Breslow & Day, 1987).\n\nWhile meta-analyses are often carried out for a specific comparison of exposure, such as current smokers vs never smokers, one often wishes to quantify the effect per unit exposure. Where exposure is measured in a consistent way in each study, and is known for each level of exposure considered in each study, standard techniques are available (described in the Methods section) to derive such trend estimates. However, where exposure may be measured in various ways, and is only defined semi-quantitatively (e.g. high, medium, low) this is not the case.\n\nHere we introduce a new, “Uniform Scale”, approach to deal with this problem. It is based on the assumption that the exposures range from 0 (least possible) to 1 (most possible), and that the N participants in any study have equally spaced exposures ranging from 1/(2N) to 1−1/(2N). By attempting always to derive an effect estimate relating to a difference of 1 unit of exposure, this “Uniform Scale” approach allows the combination of effect estimates using different measures of an underlying common exposure.\n\nWe describe how effect estimates using this “Uniform Scale” approach can be derived.\n\nWe also make available an extended version of our software to allow estimation of trend estimates, whether the exposure is quantified in standard units or expressed in the “Uniform Scale”. This extended software, now available also in R as well as in Excel and SAS, can be used for cross-sectional studies as well as for case-control and prospective studies. The new software avoids problems some users had with the original Excel spreadsheet in generating the pseudo-numbers, due to the solver routine used, a Microsoft add-in. The new Excel software is written mainly in Visual Basic, to allow matrix inversion for matrices of variable size (which is needed for the new methodology but is not readily possible using Excel formulae). Note that the extensions of the software which relate to the inclusion of trend estimates make little sense for data subdivided by level of disease, the software here being therefore essentially unchanged.\n\n\nMethods\n\nGiven a set of pseudo-numbers for the levels of exposure for a study, together with an estimate of the mean exposure for each level, it is often required to estimate the effect (and 95% CI) corresponding to a 1 unit increase in exposure. This enables the meta-analyst to produce combined estimates over studies based on effect estimates expressed for differing levels of exposure. Thus, for example, if interest is in the risk increase per cigarette smoked, one can combine these trend estimates over study, even when one study might report results by levels of, say, 0, 1–5, 6–10, 11–15, 16–20, 21–30 and 31+ cigarettes per day and another reports results by levels of 0, 1–19, 20 and 21+ cigarettes per day. Provided one can derive reasonable mean consumption estimates for each exposure level and one assumes that there is a linear relationship between dose and the logarithm of the effect estimate, these trend estimates of risk increase per cigarette per day can then be readily combined in a meta-analysis.\n\nThe methodology used for case-control and cross-sectional studies is that described by Berlin et al, (1993), together with the correction for the non-independence of results by exposure level given by Greenland & Longnecker (1992). Orsini et al. (2012) provided the modifications to be used for prospective studies. The method first derives the variance of the effect estimate for each exposure level using the width of its 95% CI. The table of pseudo-numbers is then used to estimate the correlation of pairs of results, those values then being used, together with the variance values, to estimate the covariance of each pair. The variance-covariance matrix is then inverted, and used, together with the effect estimates and dose values (mean exposure levels) to estimate beta, the coefficient of the relationship between the dose and the logarithm of the effect estimate, and its variance. Finally the values of beta and its variance are exponentiated to give the rate of increase in the effect estimate per unit increase in dose.\n\nThe methodology described above assumes that the unexposed group has a dose value of zero. If the unexposed dose is a non-zero value, this value is subtracted from each of the dose values specified. Subtracting the same value from each dose value does not change the slope of the relationship, so the estimate of beta is unaffected.\n\nIn some situations, effect estimates are presented by level of exposure where the level is merely expressed as, for example, low, medium or high, with no quantitative estimate of the extent of exposure. An example is data relating initiation of smoking in adolescents to “connectedness” (a feeling of belonging to or having affinity with a person, social group or organisation), where connectedness may be measured in various different ways, e.g. connectedness to school, connectedness to parents, or social connectedness. If these measures all relate to a common underlying scale, one could consider combining effect estimates for the various measures in a single meta-analysis. But what scale could be used?\n\nOne possible approach, and the one we suggest here, is to imagine an underlying “Uniform Scale”, where 0 indicates the least possible connectedness and 1 the most possible connectedness, with the population considered to be made up of N individuals with equally spaced scores ranging from 1/(2N) to 1−1/(2N). Thus, if there are 100 individuals in total, the individuals would have scores of 0.005, 0.015, 0.025 … 0.975, 0.985 and 0.995, with a mean score of 0.5. If there are 30 individuals in the low group, 50 in the medium group and 20 in the high group, the mean scores in the three groups would then be 0.15, 0.55 and 0.90. Or more formally, with a total of N subjects divided into k exposure groups of size N1, N2, … Nk respectively, the mean scores in the k groups would be 1/N times, respectively, N1/2, N1 + N2/2, N1 + N2 + N3/2, N1 + N2 + N3 + N4/2, and so on.\n\nThese scores are estimated from the numbers of controls for case-control studies, from the numbers at risk for prospective studies and from the total numbers (of cases and non-cases) for cross-sectional studies. The method of estimating the increase in risk per unit exposure is then identical to that described in the previous section.\n\nNote that, within the software provided, the estimated rate of increase given for the “Uniform Scale” is based on scores derived from the table of pseudo-numbers. If the user wishes to see the estimate based on scores derived from the actual distribution, these scores have to be calculated by the user, and then entered as the dose values, the estimated rate of increase then appearing as the “Dose as entered” estimate per unit exposure. The software gives the rate of increases in risk per unit exposure for both methods, so that the user can decide which method is more appropriate and so select the relevant results.\n\nSometimes results by dose may be presented in ways for which the “Uniform Scale” values can be calculated without the need to use the table of pseudo-numbers or the numbers of individuals in the study.\n\nIn some studies, the effect estimate presented relates to a comparison of two groups, one with low and one with high exposure, where the groups together cover the whole population. To include these in “Uniform Scale” meta-analysis one should square the effect estimate (and its 95% CI). To demonstrate this, assume that a proportion x has the high value, and 1−x the low value. As the mean scores for the low and high value are respectively (1−x)/2 and 1−x/2, they differ by 0.5, regardless of x, and as a linear relationship is assumed between the score and the logarithm of the effect estimate, the effect estimate should be raised to the power (1/0.5), i.e. squared.\n\nNote that where the source only provides an effect estimate for high vs low exposure, with intermediate exposures possible, application of the “Uniform Scale” methodology requires additional assumptions. Even if, for example, high and low represent the upper and lower thirds of the distribution, giving mean scores of 0.167 and 0.833, raising the effect estimate to the appropriate power, 1/(0.833–0.167) = 1.5, would assume that the dose-response relationship adequately fitted the complete data, although information on the effect for the intermediate exposure was not provided.\n\nIn other studies, the effect estimate (and CI) may be presented in relation to a one point increase in a continuous scale ranging from 0 (least possible exposure) to m (most possible exposure). Here the effect may be simply converted to our “Uniform Scale”, with a range of 0 to 1 corresponding to the difference between least and most possible exposures, by raising the effect estimate (and CI) to the mth power.\n\nWhere the effect estimate (and CIs) presented relates to a one level increase in a scale with m levels, where each level relates to a range of exposures (e.g. five levels with 1=very low, 2=low, 3=medium, 4=high and 5=very high), approximate effect estimates (and CIs) may also be obtained by mth power transformation. Thus, in the example with 5 levels and a range of 4, transforming to the 5th power is appropriate. If the levels represent m-tiles, this approximation would be exact, as a one level increase would be equivalent to an increase of 1/m in the “Uniform scale”.\n\nIn the situations described above in this section the software we provide is not required, as the meta-analyst can readily convert effect estimates (and 95% CIs) to the required “Uniform Scale” by simply raising them to the required power.\n\nIn these calculations of the “Uniform Scale” effect estimates, as illustrated in the Results section below, it is sometimes necessary to know the standard deviation (SD) of the N individual values on the “Uniform Scale”, 1, 3, 5, 7…. (2N−1), each divided by 2N. As the numbers are equally spaced, the mean is 0.5, so that Ʃx = N/2. To estimate the SD we also need Ʃx2. Since the sum of squares of the first N odd numbers is N(2N+1)(2N−1)/3, we can divide this by 4N2 to get the required value of Ʃx2. For large N, Ʃx2 = N/3. Based on the formula for the SD, it can then be shown that for large N, the SD is 1/√12 or 0.2887. This approximation is very good for the values of N usually reported for studies. Thus, for N = 50, SD = 0.2915 and for N = 100, it is 0.2901.\n\nAllowing entry of results from cross-sectional studies. The original version of the software (Hamling et al., 2008) considered data only from case-control and prospective studies. The updated software also allows entry of data from cross-sectional studies. In nearly all aspects the methodology for cross-sectional studies is identical to that for case-control studies, with ORs for case-control studies based on the relative frequency of cases and controls replaced by ORs for cross-sectional studies based on the relative frequency of those with and without the disease of interest. Exceptionally, when using the “Uniform Scale”, the calculation differs between case-control and cross-sectional studies. Scores are based on the distribution of exposure in the whole target population, which is best approximated by the distribution of the whole study population in cross-sectional studies and by the distribution of the controls for case-control studies. This is because controls in case-control studies are selected to have a distribution of exposure relevant to the whole target population.\n\nThe Excel implementation. The Excel spreadsheet described here is similar to that made available with our earlier paper (Hamling et al., 2008) except that it provides estimates of trend per unit dose, including trend based on the “Uniform Scale”.\n\nThe methodology for estimating trend involves matrix inversion. The spreadsheet needs to handle matrices of variable size, depending on the number of exposure levels included in analysis. In Excel this is not possible using formulae, so much of the new Excel code is written in Visual Basic. Visual Basic is also used for additional validity checks on the data entered.\n\nThe updated spreadsheet attempts to avoid problems encountered in the original software. The Excel software uses a Solver routine (a Microsoft add-in) when generating the table of pseudo-numbers. Updates to the Microsoft operating system meant that this Solver routine ceased to work for some users. The new version of the software avoids these problems by using methods to ensure that the Solver add-in relevant to the user’s operating system is available for use. This has been tested on several versions of the Microsoft operating system.\n\nThe updated Excel spreadsheet and its documentation can be downloaded from Zenodo RREst_trend.xlsm and RREst_trend.pdf respectively (Lee et al., 2019). Also available at that site are details of the testing of the spreadsheet: see RREst_Trend_Test_Files.zip, which contains Testing of RREst_Trend in R SAS and Excel.pdf (describing the testing carried out) and the .xlsm files (which provide the details entered and results of each test).\n\nThe Excel spreadsheet is provided in .xlsm format because it uses Visual Basic code. This file format can be accessed using Microsoft Excel 2007 and later versions.\n\nBefore opening the spreadsheet in Excel the user should ensure that the Solver add-in has been installed. Within Excel look for the Add-Ins option within Tools or Developer.\n\nThe use of macros needs to be enabled within Excel. As the spreadsheet is opened in Excel the user may be asked to confirm that they wish to continue opening a file containing macros.\n\nAs in the previous version, the spreadsheet provides drop-boxes for selecting categorization (by exposure levels or by categories of disease) and study type, the updated version allowing for cross-sectional studies as well as for case-control and prospective studies.\n\nThe actions to be taken by the user are:\n\n(1) Select categorization and study type using the drop-boxes\n\n(2) Enter the 2 × 2 table of numbers of participants. For studies categorized by exposure the rows of the table are always “unexposed” and “all exposed”, while the columns vary by study type: cases and controls for case-control studies; cases and at risk for prospective studies; and cases and non-cases for cross-sectional studies. For studies categorized by disease, the rows and columns are transposed. In this 2 × 2 table the “unexposed” is the reference category presented in the study report, while “all exposed” represents the sum of all the other categories reported. Together they represent the whole study population.\n\n(3) Enter, for each category (“exposed” level or category of disease) its name, the OR/RR estimate and its lower and upper 95% confidence limit.\n\n(4) Enter values in the contrast column. Rows given value 0 or 1 are included in analysis, rows given value -1 are excluded. In the estimation of overall risk, rows given value 0 constitute the baseline, rows given value 1 constitute the exposed.\n\n(5) Enter the dose values for the trend tests. The doses should be proportional to the amount of exposure. They are not meaningful for results categorized by disease type. Dose values for excluded rows are not used in analysis.\n\n(6) Click the Calculate button to generate the estimated numbers of subjects (the pseudo-numbers which appear in the columns to the right of the entered category details) and to produce the required results. The overall risk for the specified contrast (OR/RR and 95% CI) and the results for the heterogeneity and trend test (chi-squared and p values) are as in the earlier version of the spreadsheet. The new results are the “Trend: rate of increase in risk per unit dose” (Rate and 95% CI) giving estimates both using the dose as entered and using the “Uniform Scale”.\n\nThe data entry area and the results all appear on the left-hand side of the spreadsheet, in columns A to H. This area also provides space to enter a heading (in rows 1 and 2) and notes (in rows 54–60). Saving the spreadsheet to a relevant file name and location preserves the details of the text and data entered, and the results produced.\n\nColumns J to AF give additional information. Rows 1 to 19 give instructions to the user and notes, while rows 20 onwards give details of the underlying calculations. These include the adjusted dose values for the doses as entered (column AA) if the dose for the reference level is not zero, the dose-values using the “Uniform Scale” estimated from the pseudo-numbers (column AB) and the adjusted version of these (column AC). Adjustment simply involves subtracting the dose value for the reference level from all the other dose values.\n\nThis spreadsheet has been tested under Microsoft operating systems WIN 7, WIN 8.1 and WIN 10 using Excel 2010 and 2013. It has also been tested on MacBook using operating system Mac OS 10-13 (High Sierra) running Excel 2016.\n\nThe R implementation. This was developed as a web application using the Shiny “Web Application Framework for R” package. The application can be accessed at https://roelee.shinyapps.io/R_RRest/. The R code is available from Zenodo as the file app.R (Lee et al., 2019). Also available at that site are the method for estimating goodness of fit (described in the file Goodness of fit tests for fitted RRs.pdf) and details of the testing of the R code: see RREst_Trend_Test_Files.zip, which contains Testing of RREst_Trend in R SAS and Excel.pdf (describing the testing carried out) and the .csv files that give the input data used and the results generated in each test.\n\nThe Shiny app can be accessed in various browsers including those available for Windows, Apple and Android operating systems. The source code is provided to allow users to inspect and possibly modify the code. The code was developed in R Version 3.5.1 (2018-07-02).\n\nData entry and obtaining the required statistics are very similar in the R and the Excel implementations.\n\nIn the Shiny app, tabs at the top of the screen allow the user to “enter data for study”, see the “result for specified contrast”, see the table of “pseudo-numbers” or read the “notes” on using the application.\n\nIn “enter data for study”, the user enters first the number of exposed levels, the title and the study type. The user must then enter, in the “2x2 Table” and in “RRs, Contrasts and Doses”, the information described in items 2 to 5 of The Excel implementation above. Pressing the “solve” button on the left will then generate the pseudo-numbers and the required results.\n\nThe results include all those given in the Excel implementation. Additionally, for both trend types, the trend coefficient (the logarithm of the increase in risk per unit dose) and its standard error are shown; together with a measure of the goodness-of-fit of the trend (chi-squared value, its degrees of freedom and p value).\n\nFor a prospective study, where the pseudo-numbers of cases are Ai (i = 0, 1 … k) and the pseudo-numbers at risk are Ni (i = 0, 1 … k), the goodness-of-fit test is obtained by determining fitted numbers of cases, Fi (i = 0, 1, … k) which satisfy the formulae\n\n\n\nwhere Ri are the RR values fitted using the dose and the estimated beta value. These formulae can be solved directly and the goodness-of-fit chi-squared statistic is then derived in the usual way from the formula\n\n\n\non k-1 degrees of freedom.\n\nFor a case-control study, where the pseudo-numbers of cases are Ai (i = 0, 1 .. k) and the pseudo-numbers of controls are Bi (i = 0, 1 .. k), the goodness-of-fit test is obtained by determining fitted numbers of cases, Fi (i = 0, 1, .. k), and controls, Gi (i = 0, 1, .. k), which satisfy the formulae\n\n\n\nwhere Oi are the OR values fitted using the dose and the estimated beta value. These formulae can be solved by numerical methods (such as Newton Raphson) and the goodness-of-fit chi-squared statistic is then derived using the formula\n\n\n\non 2k-1 degrees of freedom.\n\nGoodness-of-fit testing for a cross-sectional study is equivalent to that for a case-control study, with controls replaced by non-cases.\n\nThe R implementation also allows the user easily to load data from a previously saved .csv file and to save the study data and results as a .csv file.\n\nThe Shiny application has been tested using Mozilla Firefox under Microsoft operating systems WIN 7, WIN 8.1 and WIN 10 and also using Microsoft Edge (under WIN 10) and Internet Explorer (under WIN 8.1). It has also been tested using Safari on MacBook under operating system Mac OS 10–13 (High Sierra).\n\nThe SAS implementation. The SAS implementation is provided as the macro RREst_trend.sas and is available from Zenodo (Lee et al., 2019). The documentation of the SAS implementation is also given on that website as RREst_trend SAS.pdf. Also available at that site are the method for estimating goodness of fit (described in the file Goodness of fit tests for fitted RRs.pdf) and details of the testing of the SAS code: see RREst_Trend_Test_Files.zip, which contains Testing of RREst_Trend in R SAS and Excel.pdf (describing the testing carried out) and the file SAS_RREst_Test_Results.pdf which provides the details of each test.\n\nUsers need a licenced installation of SAS in order to use the SAS code provided.\n\nThe macro has the following parameters:\n\n■ ds1 - Dataset 1, the name of the input dataset containing the OR/RR, Lower CI, Upper CI, contrast and dose values for each of the exposed levels.\n\n■ ds2 - Dataset 2, the name of the input dataset containing the 2x2 table.\n\n■ Type - Study type. Values: CC (case-control), PR (prospective), XS (cross-sectional); (default value CC).\n\n■ levels - How the study data is categorised. Values: EX (by exposure), DI (by disease); (default value EX).\n\n■ out - Name of the output dataset that will hold the pseudo-numbers (default _RREst_).\n\n■ alpha - Error probability used for the confidence intervals of the data entered (default value 0.05, equivalent to 95% CI).\n\n■ trend - Report the trend tests? Values: 1 = Yes, 0 = No (default value 0).\n\n■ details - Output the detailed results (details of each iteration and the final P’ and Z’ values)? Values: 1 = Yes, 0 = No (default value 0).\n\n■ grid - Step size (out of 0–1) that should be used for finding a starting point for the iterative process (default value 0.01).\n\n■ ini_beta - Starting point for the iterative process (default value: use the “grid” parameter’s starting point).\n\nThe first two of these parameters must be specified. If they are entered as the first two parameters, the parameter names (ds1 and ds2) are assumed and so need not be entered. The other parameters are optional. They are specified using the format parameter name = value. If not specified, the default value will be used. For example,\n\n\n\nHere the macro is called only specifying the two input datasets containing, respectively, the details of the exposure categories and the 2x2 table. All other parameters take their default values, including study type case-control with data presented by levels of exposure and giving the pseudo-numbers dataset the name _RREst. If the study is prospective, the SAS macro could be called using\n\n\n\nDataset 1 (ds1) should contain the details of each exposure level (or disease type) that makes up the study population, including the unexposed level. The fields within the dataset should be named as follows:\n\n■ level - Equivalent to the Category column in the Excel implementation, giving a description of the exposure category (or disease type).\n\n■ Est - OR/RR value. Ignored for the unexposed level.\n\n■ lower - Lower confidence limit. Ignored for the unexposed level.\n\n■ upper - Upper confidence limit. Ignored for the unexposed level.\n\n■ dose - Mean exposure level (trend coefficients). Equivalent to the Dose column in the Excel implementation. If not included, dose values are assumed to be 0, 1, 2, and so on.\n\nIn addition, one or more contrast fields should be entered. These are equivalent to the Contrast column in the Excel implementation. Results will be generated for each contrast specified. Exceptionally, these fields can be given any name.\n\nDataset 2 (ds2) is equivalent to the 2x2 table in the Excel implementation. It must contain two fields, their names depending on study type and categorisation:\n\n■ Any study categorised by disease type - “Exposed” and “Unexposed”\n\n■ Case-control study, by exposure - “Cases” and “Controls”\n\n■ Prospective study, by exposure - “Cases” and “At_Risk”\n\n■ Cross-sectional, by exposure - Cases” and “Non-cases”\n\nIn order to contain the 2x2 table values, it needs to have two data rows.\n\nThe output from the SAS macro is presented in the output window. This output includes the trend results using the “Uniform Scale” and the goodness-of-fit results as described above for the R implementation. The output is also written to output files, as described in the detailed documentation (available from Zenodo, file RREst_trend SAS.pdf) (Lee et al., 2019).\n\nThis SAS code has been tested using SAS 9.4 (64 bit) under Microsoft operating system WIN 7 and using SAS 9.4 (32 bit) under WIN 10.\n\n\nResults\n\nIn this section we give examples of applying the methodology. We do this using the results presented in two papers, one a report of smoking habits and lung cancer risk in Norway (Engeland et al., 1996) and the other a report on initiation of tobacco use among adolescents in the USA (Karcher & Finn, 2005).\n\nWe used the first of these (Engeland et al., 1996) to demonstrate using the method to estimate the effect per unit dose of exposure. This paper reports several dose measures, including age of starting smoking and intensity of pipe smoking. We considered the dose measure intensity of cigarette smoking, measured as the number of cigarettes habitually smoked per day. The outcome of interest was lung cancer of any type. The paper reports a study of 8,905 men born between 1893 and 1927 who were followed up for 28 years. It provides a risk assessment with confidence interval for each of five categories of number of cigarettes smoked per day. We use these results to estimate the increase in risk associated with the consumption of one extra cigarette per day.\n\nThe second paper (Karcher & Finn, 2005) reports a cross-sectional study of 303 middle and high school students from a rural town in Midwest USA. It used a Measure of Adolescent Connectedness (MAC) instrument to assess the adolescents’ degree of caring for and involvement in specific relationships. This instrument included an assessment of parental connectedness, reported as low, medium and high connectedness. The paper reports odds ratios for experimental smoking by levels of parental connectedness. These levels of connectedness cannot be assessed as a numerical dose, but the “Uniform Scale” approach can be used.\n\nAll data input are available as underlying data (Lee et al., 2019).\n\nIdentifying the data to be used. In the cohort study on lung cancer risk in Norwegian men and women, Engeland et al. (1996) presented data in men on the numbers of lung cancer cases and of person-years for seven groups – never smokers (the reference group), former smokers and current smokers of, respectively, 1–4, 5–9, 10–14, 15–19 and 20+ cigarettes per day. Dividing the numbers of person-years by the number of years of follow-up (28) to give an approximate indicator of the numbers at risk, and combining the results for all the exposed groups (the last six groups), the numbers of cases were 27 in never and 306 in ever smokers, while the corresponding numbers at risk were 2,097 and 6,017. These numbers were used to populate the 2x2 table of numbers in the Excel program. Note that this table is only used as a starting point to the iterative process for estimating the pseudo-numbers so does not have to be precise.\n\nThe estimation of the pseudo-numbers also requires the adjusted RRs (95% CIs) for the seven groups, which are given in the paper as, respectively, 1.0, 1.3 (0.8 to 2.2), 1.4 (0.6 to 3.7), 4.1 (1.7 to 10.0), 7.0 (2.9 to 17.0), 11.0 (4.2 to 28.0) and 15.0 (6.1 to 37.0). For dose assessment the midpoint consumption level for each category of current smokers are also needed, which we derived using the standard distribution described by Fry & Lee (2000) as 2.5, 6.5, 10.88, 15.83 and 26.03.\n\nAs shown in Table 1, we entered the relevant data into the Excel spreadsheet. Although we subsequently excluded the result for former smokers from analysis, the whole study population was counted in the 2x2 table of numbers of participants and details of each exposure level (never smokers, former smokers and the five levels of smoking intensity) were entered because the estimation of pseudo-numbers would then be based on all the information we have about the study. For this example study we have available the numbers of participants (cases and at risk) for each of the exposure levels. Often a study will report only a summary of the numbers of participants such as the totals exposed and unexposed or the overall total numbers of participants and the proportions exposed (cases and at risk). These summary details are sufficient to allow the method to be used. The results shown indicate the following:\n\na The reference group in the study population: the never smokers\n\nb All non-reference participants in the study population: the sum of values for former smokers and the five groups of current smokers\n\nc Approximate indicators – see text\n\nd These should be 0 or 1 for the levels to be included in the analysis. The Overall risk result compares the exposed group levels (value 1) with the reference group level(s) (value 0).\n\ne We have valid dose information for the exposure categories so results based on the “Uniform Scale” are ignored.\n\nTable of pseudo-numbers. This table is consistent with the input RR and 95% CI values in that using the standard formulae for estimating each RR (CI) from a 2 x k table will generate the results input. It is notable that the numbers of cases in smokers are much lower than the original numbers cited in the paper. This is partly due to the effects of adjustment, but also because the original numbers given in the paper are not consistent with the varying widths of the 95% CIs, which are much narrower for former smokers than for each level of current smoking.\n\nOverall risk for the specified contrast. This calculation is identical to that provided in the original software. Given the contrast values chosen of 0, −1, 1, 1, 1, 1 and 1 the overall risk value is an estimate of the RR in all current smokers combined (contrast value 1) relative to never smokers (contrast value 0) with former smokers excluded (contrast value -1). The contrast values are not relevant to the trend analysis, except that the selection of −1 for former smokers causes that exposure level to be excluded from analysis, so the trend analysis is based only on the data for never and current smokers.\n\nHeterogeneity and trend (Breslow, 1980). These values are identical to those given in the original software, and confirm that the trend is very highly significant, and also that the trend explains the major part of the heterogeneity between levels.\n\nTrend: Rate of increase in risk per unit dose. Using the dose values input, the estimated RR (95% CI) is 1.1228 (1.0879 to 1.1588). This 12.28% increase per unit dose implies that the fitted risk for the five current smoking groups is 1.34, 2.12, 3.52, 6.25 and 20.26, as compared with the input values of 1.4, 4.1, 7, 11 and 15. As discussed elsewhere (Fry et al., 2013), the shape of the dose-response relationship of lung cancer risk to amount smoked may be better fitted by alternative non-linear models.\n\nIn the cross-sectional study relating connectedness to experimental smoking among rural youth, Karcher & Finn (2005) reported that there were 135 experimental smokers, 43 with low parental connectedness, 54 with medium connectedness and 38 with high connectedness. As these numbers formed respectively 55%, 50% and 32% of the numbers of youths at each level of connectedness, we could estimate that the numbers who were not experimental smokers were 35 for low, 54 for medium and 81 for high parental connectedness, giving totals by level of 78, 108 and 119.\n\nThe exposure scale is qualitative so the method of calculating effect estimates using actual dose values cannot be used. However, our “Uniform Scale” methodology can be used to give a result that is comparable with those for other measures of connectedness in the same or a similar population.\n\nThis study provides the numbers of participants in each exposure level (78, 108 and 119 for low, medium and high connectedness respectively) so, as a demonstration of the method, we can calculate the “Uniform Scale” scores without using the software. Calculating N1/2, N1 + N2/2 and N1 + N2 + N3/2, gives the values of 39, 132 and 245.5. Scaling these to the range 0–1 is achieved by dividing by the total number of participants (305), giving scores on the “Uniform Scale” of 0.1279, 0.4328 and 0.8049.\n\nThe software uses the same method to derive “Uniform Scale” scores but bases them not on the actual numbers of participant by exposure level (which are often not available in a study report) but instead on the table of pseudo-numbers. Using this example we can compare the scores and the related trend results estimated using the pseudo-numbers with those based on the actual numbers of participants (above).\n\nThe authors also presented adjusted ORs (95% CIs) of 1.26 (0.69 to 2.27) for low vs medium parental connectedness and of 2.55 (1.40 to 4.66) for low vs high parental connectedness. As we wish to estimate ORs relative to low, we inverted these to get 0.7937 (0.4405 to 1.4493) for medium vs low and 0.3922 (0.2146 to 0.7143) for high vs low.\n\nAs shown in Table 2, we entered the relevant data into the Excel spreadsheet. In the Dose column we entered the “Uniform Scale” scores calculated above (based on the actual numbers of participants) so that the two sets of trend results generated both related to the “Uniform Scale” scores, one using the actual numbers of participants and the other based on the pseudo-numbers. Papers do not usually give numbers of participants in each exposure level and so “Uniform Scale” dose values based on actual numbers of participants cannot be calculated and entered. In these circumstances the results based on the Dose column values (Trend “Using the doses as entered”) should be ignored.\n\na The reference group in the study population: low parental connectedness\n\nb All non-reference participants in the study population: medium + high parental connectedness\n\nc These should be 0 or 1 for the levels to be included in the analysis. The Overall risk result compares the exposed group levels (value 1) with the reference group level(s) (value 0).\n\nd In this example these have been set to the “Uniform Scale” values calculated using the actual numbers of participants in each level\n\nThe results include the table of pseudo-numbers. This table is again consistent with the input OR and 95% CI values. The numbers are slightly lower than the original numbers, due to the increase in variance following adjustment.\n\nAnother result presented is the overall risk for the specified contrast. Given the input contrast values of 0, 1 and 1 the resulting OR relates to the pairwise comparison of medium and high combined to low. The contrast values are not relevant to the trend analysis except that entering a contrast value of −1 would have caused the exposure level to be excluded from analysis, though not from the calculation of the pseudo-numbers.\n\nThe output also shows the results of the trend analysis – the rate of increase in risk per unit dose. Using the scores calculated above, based on the actual numbers, the estimated OR (95% CI) for a 1 unit difference in exposure (most exposed compared with least possible exposure) is 0.2382 (0.0990 to 0.5730). This is very similar to the estimate based on the pseudo-numbers, of 0.2388 (0.0994 to 0.5738), because the “Uniform Scale” scores based on the pseudo-numbers are very similar to those based on the actual numbers. Note that the estimated reduction in risk is larger than that for the high vs low comparison (OR 0.3922, 95% CI 0.2146 to 0.7143), as that comparison is only based on an estimated difference in exposure of about 0.68 units rather than being based on a difference of 1 unit.\n\nIn addition to the example above, it is useful to give some other examples where the nature of the information presented needs particular consideration in order to provide effect estimates in the “Uniform Scale”.\n\nOne example is the study by Lloyd-Richardson et al. (2002), which reports results from a cross-sectional study based on analyses of 19 818 adolescents, of whom 10 924 had at least experimented with smoking. The authors present an OR (CI) of 1.16 (1.03–1.30) for low school connectedness, assessed using an eight item scale, the number of levels per item not being given. The authors note that, in coding the data, the variable was standardized by “subtracting off the median and dividing them by the distance between the median and the third quartile”. As the median corresponds to a mean score of 0.5 and the third quartile to a mean score of 0.625 (midway between 0.5 and 0.75) this suggests the difference in mean score is 0.125, so that the OR (CI) should be raised to the eighth power (1/0.125 = 8) to give the required result of 3.28 (1.27–8.16).\n\nAnother example is the study by Simons-Morton & Haynie (2003) in which 973 students completed surveys at the beginning and end of the sixth grade. Their Table 4 gives an OR for high v low social competence of 0.71 (0.52–0.98). The authors note that social competence is measured using eight items, with a mean of 22.37 (SD 5.39). It is unclear how many levels there are for each item, though possibly four given the mean. Nor is it clear whether high v low is just a simple breakdown of the population into two groups, in which case the OR (CI) should be squared, as noted above, to give 0.50 (0.27–0.96). Alternatively, if high is the highest quartile, and low the lowest quartile, one is comparing groups with mean scores of 0.875 and 0.125, a difference of 0.75 so that the OR (CIs) should be raised to the power of 1/0.75 = 4/3, giving a result of 0.63 (0.42–0.97). More information would be needed before this study could be included in any meta-analysis.\n\nFinally we consider the study by Kandel et al. (2004) who compared smoking onset factors in 5,374 adolescents who had never smoked based on data from the National Longitudinal Study. They report ORs of 0.59 (0.45–0.77) for positive scholastic attitudes and 0.93 (0.75–1.16) for parent-child connectedness. For positive scholastic attitudes they averaged four 5-point items, so presumably the OR related to a 1 point difference in a scale that can vary by 4 points. This indicates that one should power up the 0.59 (0.45–0.77) by 5 to give 0.07 (0.02–0.27). For parent connectedness the authors refer to a 13 item scale, but state neither the number of points in each scale nor whether the values for individual items were summed or averaged. The authors refer to Resnick et al. (1997) as using this 13-item scale, that paper stating that this variable was standardized “to a mean of 0 and an SD of 1”, so that “parameter estimates can be interpreted as standardized B”. Assuming that the paper considered here did the same, and noting that, as derived earlier, the standard deviation of our “Uniform Scale” is approximated by 1/√12 = 0.2887 for large N, one needs to power the OR up by √12 to give the units we require. This gives an OR for parent-child connectedness of 0.78 (0.37–1.67).\n\nNote that in all three of these last examples, the new software is not required, the user only having to raise reported effect estimates to the appropriate power to obtain the required estimate, corresponding to the difference in risk between the most and least possible exposures.\n\nNote also, that when conducting meta-analyses it is important to be sure that the effect estimates are calculated in the same direction. If some give results for high v low and some for low v high, it will be necessary to invert the estimates as appropriate to ensure combinability.\n\nIt is of interest to see what results would have been obtained in the example shown in Table 1 had we not had estimates of the dose for never smokers and for current smokers by amount smoked, but instead used the “Uniform Scale” methodology to estimate doses for each level based on the distribution of the at risk population. Here the OR (CI) is estimated as 29.5493 (11.0943 to 78.7039) as compared with the original trend estimate of 1.1228 (1.0879 to 1.1588). The OR of 29.5493 is equal to 1.1228 raised to the 29.23th power. As the original estimate is per cigarette per day, this would imply that the “Uniform Scale” relates to a full range of 29.23 cigarettes per day. While no doubt some smokers smoke more cigarettes per day, this result seems not to be implausible given the original calculation was based on dose means, with that for the heaviest smoking group estimated as 26.03 cigarettes per day.\n\n\nDiscussion/conclusions\n\nSince we published our original paper in 2008 (Hamling et al., 2008), we have carried out numerous meta-analyses making extensive use of the software (e.g. (Forey et al., 2011; Lee et al., 2012; Lee et al., 2017; Lee et al., 2016a; Lee et al., 2016b; Lee & Hamling, 2009; Lee & Hamling, 2016)). We have also carried out dose-response meta-analyses (Fry et al., 2013), though we have only recently updated the Excel software. While the methodology has proved useful to us and to other researchers in situations where the raw data for a study are not available, there are some difficulties in applying it. These include presentation of the original data to insufficient accuracy, not having available the required 2x2 table (e.g. cases/controls x unexposed/exposed for a case-control study) and so having to use approximate estimates; and sometimes finding that the estimation of the pseudo-numbers does not converge to a solution, even after using different starting points for the iteration process. Nevertheless, the method gave what appeared plausible estimates in many practical applications.\n\nExtending the software to estimate increases in risk per unit of exposure does bring a few additional problems. One is the difficulty in obtaining an estimate of the midpoint exposure for data grouped by ranges of exposure, especially when the highest range is open-ended. Another is that the trend estimation assumes an underlying dose-response shape that may not necessarily apply. Nevertheless, the extensions to the software to include trend estimation does provide the meta-analyst with a useful tool for combining dose-response results from different studies that use varying ways of grouping dose.\n\nThe extension of the software to use the “Uniform Scale” also allows the meta-analyst to attempt combination of results from studies using qualitative rather than quantitative estimates of exposure, possibly derived using a variety of measures of some underlying exposure. By attempting to derive an effect estimate for the range from the most exposed possible (with score 1) to the least exposed possible (with score 0), the meta-analyst is given a way of combining results on a consistent basis. Clearly, the underlying assumption - that the study population is made up of individuals with successively increasing exposure by an equal amount - is dubious, but we nevertheless feel that the method is useful. Other approaches, but with a different specification of the distribution of exposure, may well be possible, but we have not investigated these so far.\n\n\nData availability\n\nZenodo: Software for use in meta-analysis, providing Effect estimates per unit of exposure, including the Uniform scale. http://doi.org/10.5281/zenodo.3582481 (Lee et al., 2019)\n\nThis project contains the following underlying data:\n\n■ RREst_Trend_Test_Files.zip (various files describing the testing carried out, including .xlsm files used in testing the Excel version and .pdf files describing the R and SAS testing: the Methods section above names the files relevant to the testing of each implementation).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nSoftware availability\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.3582481 (Lee et al., 2019)\n\nLicense: Creative Commons Attribution 4.0 International license (CC-BY 4.0)\n\nThis contains the following files:\n\napp.R (the code for the R implementation)\n\nRREst_trend.SAS (the SAS implementation)\n\nRREst_trend.xlsm (the Excel implementation)\n\nRREst_trend.pdf (the Excel implementation documentation)\n\nRREst_trend SAS.pdf (the SAS implementation documentation)\n\nGoodness of fit tests for fitted RRs.pdf (a document describing how the goodness-of-fit statistics are calculated)", "appendix": "Acknowledgements\n\nThe authors thank Mrs. Yvonne Cooper and Mrs. Diana Morris for typing the various drafts of this paper, and Mr. John Hamling for assistance with software testing.\n\n\nReferences\n\nBerlin JA, Longnecker MP, Greenland S: Meta-analysis of epidemiologic dose-response data. Epidemiology. 1993; 4(3): 218–228. PubMed Abstract \| Publisher Full Text\n\nBreslow NE, Day NE: Statistical methods in cancer research. Volume I - The analysis of case-control studies. IARC Sci Publ. 1980; (32): 5–338. PubMed Abstract\n\nBreslow NE, Day NE: Statistical methods in cancer research. Volume II--The design and analysis of cohort studies. IARC Sci Publ. 1987; (82): 1–406. PubMed Abstract\n\nEngeland A, Haldorsen T, Andersen A, et al.: The impact of smoking habits on lung cancer risk: 28 years' observation of 26,000 Norwegian men and women. Cancer Causes Control. 1996; 7(3): 366–376. PubMed Abstract \| Publisher Full Text\n\nForey BA, Thornton AJ, Lee PN: Systematic review with meta-analysis of the epidemiological evidence relating smoking to COPD, chronic bronchitis and emphysema. BMC Pulm Med. 2011; 11: 36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFry JS, Lee PN: Revisiting the association between environmental tobacco smoke exposure and lung cancer risk. I. The dose-response relationship with amount and duration of smoking by the husband. Indoor Built Environ. 2000; 9(6): 303–316. Publisher Full Text\n\nFry JS, Lee PN, Forey BA, et al.: Dose-response relationship of lung cancer to amount smoked, duration and age starting. World J Metaanal. 2013; 1(2): 57–77. Publisher Full Text\n\nGreenland S, Longnecker MP: Methods for trend estimation from summarized dose-response data, with applications to meta-analysis. Am J Epidemiol. 1992; 135(11): 1301–1309. PubMed Abstract \| Publisher Full Text\n\nHamling J, Lee P, Weitkunat R, et al.: Facilitating meta-analyses by deriving relative effect and precision estimates for alternative comparisons from a set of estimates presented by exposure level or disease category. Stat Med. 2008; 27(7): 954–970. PubMed Abstract \| Publisher Full Text\n\nKandel DB, Kiros GE, Schaffran C, et al.: Racial/ethnic differences in cigarette smoking initiation and progression to daily smoking: a multilevel analysis. Am J Public Health. 2004; 94(1): 128–135. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKarcher MJ, Finn L: How connectedness contributes to experimental smoking among rural youth: developmental and ecological analyses. J Prim Prev. 2005; 26(1): 25–36. PubMed Abstract \| Publisher Full Text\n\nLee PN, Forey BA, Coombs KJ: Systematic review with meta-analysis of the epidemiological evidence in the 1900s relating smoking to lung cancer. BMC Cancer. 2012; 12: 385. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee PN, Forey BA, Hamling JS, et al.: Environmental tobacco smoke exposure and heart disease: A systematic review. World J Metaanal. 2017; 5(2): 14–40. Publisher Full Text\n\nLee PN, Fry JS, Forey B, et al.: Environmental tobacco smoke exposure and lung cancer: a systematic review. World J Metaanal. 2016a; 4(2): 10–43. Publisher Full Text\n\nLee PN, Hamling JS, Fry JS, et al.: Software for use in meta-analysis, providing Effect estimates per unit of exposure, including the Uniform scale (Version 1.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3582481\n\nLee PN, Hamling J: Systematic review of the relation between smokeless tobacco and cancer in Europe and North America. BMC Med. 2009; 7: 36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee PN, Hamling JS: Environmental tobacco smoke exposure and risk of breast cancer in nonsmoking women. An updated review and meta-analysis. Inhal Toxicol. 2016; 28(10): 431–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee PN, Thornton AJ, Hamling JS: Epidemiological evidence on environmental tobacco smoke and cancers other than lung or breast. Regul Toxicol Pharmacol. 2016b; 80: 134–163. PubMed Abstract \| Publisher Full Text\n\nLloyd-Richardson EE, Papandonatos G, Kazura A, et al.: Differentiating stages of smoking intensity among adolescents: stage-specific psychological and social influences. J Consult Clin Psychol. 2002; 70(4): 998–1009. PubMed Abstract \| Publisher Full Text\n\nOrsini N, Li R, Wolk A, et al.: Meta-analysis for linear and nonlinear dose-response relations: examples, an evaluation of approximations, and software. Am J Epidemiol. 2012; 175(1): 66–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nResnick MD, Bearman PS, Blum RW, et al.: Protecting adolescents from harm. Findings from the National Longitudinal Study on Adolescent Health. JAMA. 1997; 278(10): 823–32. PubMed Abstract \| Publisher Full Text\n\nSimons-Morton BG, Haynie DL: Psychosocial predictors of increased smoking stage among sixth graders. Am J Health Behav. 2003; 27(6): 592–602. PubMed Abstract \| Publisher Full Text" }	[ { "id": "58931", "date": "11 Feb 2020", "name": "Chang Xu", "expertise": [ "Reviewer Expertise Evidence synthesis", "dose-response meta-analysis." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nLee et al. have presented an interesting and important work for estimating the missing data in dose-response meta-analysis. It is so appreciated for these authors for their contribution in this area. I have some further comments that hope will be helpful for the authors.\nThe background seems not well introduced. In dose-response meta-analysis, the non-reference effect estimates were correlated, when combining the dose-response relationship and taking into account the study correlation is essential for a BLUE estimation. And based on the GLST method, the group size information (cases, non-cases) were generally used to estimate the correlation. And further, some of the studies failed to provide such group size information that make it hard to get an estimation. Therefore, this article and previous one by Hamling are all an attempt to solve this problem. I believe a clear introduction for this could help readers get a better understanding.\n\nThe missing information about the group sizes could be an important issue for the GLST framework, as it uses GLS estimation which requires the co-variance matrix. There is another framework based on the robust-error to deal with the correlation, which was first proposed by Hedges et al.1, and has been introduced in dose-response meta-analysis (called robust error meta-regression method, REMR) by Suhail et al. And based on REMR framework, the group size information are no longer needed2. The authors should discuss it.\n\nThe updated software allows the cross-sectional study. This is an important and useful update. In my experience, I noticed that many systematic reviewers combine the cross-sectional study and cohort/case-control study together. However, this is not true. I suggest the authors add 1-2 sentences to the difference of cross-sectional study to the other two and remind the readers to avoid the problem.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "11289", "date": "04 Apr 2024", "name": "Peter Lee", "role": "Author Response", "response": "I thank the reviewer for his kind comments. However have decided not to follow his specific suggestions for amending the paper." } ] } ]	1	https://f1000research.com/articles/9-33
https://f1000research.com/articles/8-145/v1	04 Feb 19	{ "type": "Software Tool Article", "title": "Biobtree: A tool to search, map and visualize bioinformatics identifiers and special keywords", "authors": [ "Tamer Gur" ], "abstract": "Due to their nature, bioinformatics datasets are often closely related to each other. For this reason, search, mapping and visualization of these relations are often performed manually or programmatically via identifiers or special keywords such as gene symbols. Although various tools exist for these situations, the growing volume of bioinformatics datasets, emerging new software tools and approaches motivates new solutions. To provide a new tool for these current cases, I present the Biobtree bioinformatics tool. Biobtree effectively fetches and indexes identifiers and special keywords with their related identifiers from supported datasets, optionally with user pre-defined datasets and provides a web interface, web services and direct B+ tree data structure based single uniform database output. Biobtree can handle billions of identifiers and runs via a single executable file with no installation and dependency required. It also aims to provide a relatively small codebase for easy maintenance, addition of new features and extension to larger datasets. Biobtree is available to download from GitHub.", "keywords": [ "bioinformatics", "identifiers", "search", "mapping", "visualization" ], "content": "Introduction\n\nBioinformatics datasets often consist of entries, where each entry is represented by unique identifier. Depending on the dataset, each entry contains various types of information such as sequence data, biological function, chemical structure or literature reference etc. In addition, entries often contain cross-referencing information to other dataset entries via identifiers. Let’s take as an example entry the proto-oncogene vav protein in humans, which is encoded by the VAV1 gene. If we display this protein on the UniProt website we see cross references to many other datasets. These cross references represent relations of datasets with each other. Various tools exist to deal with such data; however, the growing volume of bioinformatics datasets, emerging new software tools and analysis approaches motivates new solutions. Biobtree1 presented herein is capable of improved and rapid processing of large numbers of unique identifiers of entries and related identifiers that are specified via cross-reference data.\n\nIn some datasets, in addition to unique identifiers there is information that is strongly related to entries but not necessarily unique for each entry. Species names or UniProt secondary accessions are example of this type. Information that is strongly related to the entries but not necessarily unique is a second data source for Biobtree. In Biobtree, these types are called special keywords and each of these can be related to multiple entries among the datasets.\n\nBiobtree retrieves all these identifiers, related identifiers and special keywords from various bioinformatics resources and stores it in a single database. The data resources currently used are ChEBI2, HGNC3, HMDB4, InterPro5, Europe PMC6 and UniProt7. Table 1 shows details of these datasets.\n\nBased on stored data, Biobtree provides search, map and visualization functionalities via provided web services or a web interface. For instance, all the UniProt proteins entries belonging to a gene name, or, all Ensembl8 genome transcripts identifiers and ENA9 sequence identifiers that map to a protein identifier can be accessed. These relations, determined via identifiers, are stored bidirectionally so all actions can also be done in the opposite way.\n\nBiobtree is managed from a single executable file for each major operating system without requiring any installation or compilation. As a database, Biobtree uses a B+ tree data structure based LMDB key value store. LMDB provides fast batch inserts and reads and allows effective operation on a large number of records. LMDB is embedded into Biobtree’s executable binary code so it does not require a separate installation.\n\n\nMethods\n\nBiobtree1 has been implemented in GO programming language. To use C programming language based LMDB in Biobtree GO environment lmdb-go binding library has been used. To implement the Web interface Javascript programming language, Vue and Bulma web frameworks has been used. Biobtree workflow consists of three phases, which will be explained in later sections. These phases are named update, generate and web and are controlled by a Biobtree command line interface (CLI)\n\n\nUpdate phase\n\nThe purpose of the update phase is to retrieve dataset identifiers and special keywords from remote servers or a local disk and produce files that contains identifiers and special keywords with their referred identifiers as keys and values in a sorted order. It is essential that the produced files are sorted to make fast batch inserts to LMDB database in the next phase. The updating phase is started via the Biobtree CLI with the update command. For example, the following command starts the update phase for the hgnc dataset.\n\n\n\nUpdating reads selected datasets as a stream and saves Biobtree-related data in a series of files. An advantage of reading dataset as streams is that it does not require fully downloading the dataset to the local disk. Datasets can have different formats like XML, JSON, TSV or CSV. Biobtree has specialized parsers for each dataset and parses them to produce its output files. When Biobtree runs the first time it retrieves its configuration, license and web interface files from the source code repository. Configuration files contain Biobtree runtime settings and dataset definitions.\n\nUser data can be integrated to Biobtree. This feature creates an alternative for data providers to serve their data. Data should be gzipped and in an xml format compliant with UniProt xml schema definition. After the file path of the data is configured in a Biobtree configuration file, updating starts similarly:\n\n\n\nBiobtree supports executing the update phase over multiple computers. This is useful when it is necessary to use multiple computer processors at the same time such as with large datasets. The following two commands can be run on different computers with additional idx argument to guarantee that the produced files have unique names.\n\n\n\nAlthough Biobtree supports the updating phase occurring over multiple computers, for the next phase all the produced files have to be in a single location.\n\n\nGenerate phase\n\nThe purpose of this phase is to merge all the files produced in the update phase by keeping the sorted order and generate the final key and values in the generated LMDB database. Keys consist of identifiers and special keywords and values are identifiers that are referred to by these keys with their dataset information. If the values size for each key are above a certain threshold they are saved in pages. The following command starts the generate phase.\n\n\n\nThe generated database output is used in next web phase but it can be also used directly. Example source codes for using the database directly can be found in the project github page.\n\n\nWeb phase\n\nThe purpose of this phase is to provide web services and a web interface via the produced output of the generate phase. The following command starts web phase\n\n\n\nWith this command the Biobtree web server is started instantly, serving the REST, gRPC and web interface services.\n\nTo make queries in the produced database, Biobtree provides RESTful endpoints with json-formatted responses. For example, each dataset has a unique identifier and other meta information like name, url template, etc. Dataset-unique identifiers are used in all the services to distinguish the dataset. These meta information is retrieved via the following endpoint:\n\nhttp://localhost:8888/ws/meta\n\nThe following are used to query single or multiple identifiers or special keywords:\n\nhttp://localhost:8888/ws/?idlist=vav_human\n\nhttp://localhost:8888/ws/?idlist=vav_human,tpi1,brca2\n\nTo make a paging query for a certain identifier:\n\nhttp://localhost:8888/ws/?id=vav_human&dataset=1&page=1\n\nTo make a filtering query based on a dataset:\n\nhttp://localhost:8888/ws/?id=vav_human&dataset=1&filters=102\n\nTo make a paging query with active filtering:\n\nhttp://localhost:8888/ws/?id=vav_human&dataset=1&filters=102&page=1\n\nRESTful services are often json-based and are very convenient in json-based applications. But for non–json-based applications, it requires an extra process of serialization and deserialization. To address this, Biobtree provides a gRPC service with same functionality as its RESTful service. Sample codes for using gRPC in different languages can be found on the project github page. The following is snippet of Biobtree gRPC service definitions:\n\n\n\nThe Web interface allow user to visualize the produced database via the RESTful service. Once the web phase is started it is accessed via the browser from the following address:\n\nhttp://localhost:8888/ui\n\nThe Web interface provides searching of multiple identifiers and special keywords, visualizing and filtering results, and executing bulk queries. On the result page for each result, it provides a url to access the main website where the data are originally produced. Figure 1 and Figure 2 show the main and result page of the web interface.\n\n\nOperation\n\nBiobtree executable file is available from GitHub page for Windows, MacOS and Linux operating systems. For default datasets and configuration Biobtree uses up to 4 GB of memory. For large datasets it is advised to use computer which has large RAM space such as 16 GB to speed up finishing update and generate phases. RAM usage for these phases can be managed from configuration file via kvgenChunkSize and batchSize variables. For CPU Biobtree uses all available CPU powers when it needed. This default behaviour can be restricted via CLI with maxcpu argument.\n\n\nBenchmarks\n\nSoftware benchmarks should often be taken with a grain of salt, especially where the input data is large, because benchmarks results can be affected by many factors like the processor, operating system, storage, network speed, input data, application parameters etc. Considering these, the purpose of Biobtree benchmarks is mainly to show overall capabilities and resource usage of Biobtree. Table 2 shows the benchmark details and results. Benchmarks were primarily computed at DigitalOcean London datacentres using their CPU optimized droplets with block storage volumes. Hundred thousand sample query which used in the benchmarks can be found on the project GitHub page.\n\n\nDiscussion\n\nThe benchmarks show that Biobtree1 has produced LMDB database output in relatively acceptable times. Let’s discuss how Biobtree behaves if UniProt provides tens of times larger data. Clearly, more disk space would have been needed.\n\nIf enough disk space is provided, the next obstacle would have happened during the update phase, because currently UniProt provides a single gzip compressed file for each dataset and Biobtree reads each file as a stream from the beginning to end. Gzip does not allow a random access to a file unless there are checkpoints defined. This characteristic of gzip prevents the processing of a single large file in a split manner and utilizes more computing resources if available. Two solutions can address the issue. The first could be for UniProt to allow its datasets to be capable of parallel processing, like splitting and compressing files inside a tar archive. The second solution would be to implement a new functionality in Biobtree and save and decompress these files to local disk and make parallel access to decompressed files.\n\nA further obstacle would have happened during the generate phase, since we would have obtained more files from update phase. The generate phase struggles to merge all these files and could cause much longer output generation times. To address this obstacle, a new phase could be implemented that runs before the generate phase and merges files coming from the update phase.\n\n\nLimitations\n\nAlthough duplicate values for each key are discarded during the update phase for each dataset, the generate phase could rarely produce duplicate values. The user needs to discard these duplicate records manually if these are created. Another limitation is when querying the special keywords, they need to be fully specified including all space characters.\n\n\nFuture work\n\nThe limitations can be addressed and new functionalities added in the future. For instance, different bioinformatics datasets like Ensembl8 or ENA9 can be integrated. Another feature would be sorting result values based on a certain criterion.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nSoftware availability\n\nAll source codes and binaries available at: https://www.github.com/tamerh/biobtree.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.25470471.\n\nLicense: BSD 3-Clause \"New\" or \"Revised\" license.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nGür T: tamerh/biobtree: biobtree v1.0.0-rc2 (Version v1.0.0-rc2). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.2547047\n\nHastings J, Owen G, Dekker A, et al.: ChEBI in 2016: Improved services and an expanding collection of metabolites. Nucleic Acids Res. 2016; 44(D1): D1214–D1219. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYates B, Braschi B, Gray KA, et al.: Genenames.org: the HGNC and VGNC resources in 2017. Nucleic Acids Res. 2017; 45(D1): D619–625. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWishart DS, Feunang YD, Marcu A, et al.: HMDB 4.0: the human metabolome database for 2018. Nucleic Acids Res. 2018; 46(D1): D608–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMitchell AL, Attwood TK, Babbitt PC, et al.: InterPro in 2019: improving coverage, classification and access to protein sequence annotations. Nucleic Acids Res. 2019; 47(D1): D351–D360. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEurope PMC Consortium: Europe PMC: a full-text literature database for the life sciences and platform for innovation. Nucleic Acids Res. 2015; 43(Database issue): D1042–D1048. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe UniProt Consortium: UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. 2019; 47(D1): D506–D515. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZerbino DR, Achuthan P, Akanni W, et al.: Ensembl 2018. Nucleic Acids Res. 2018; 46(D1): D754–D761. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarrison PW, Alako B, Amid C, et al.: The European Nucleotide Archive in 2018. Nucleic Acids Res. 2019; 47(D1): D84–D88. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "45074", "date": "07 Mar 2019", "name": "Maxim N. Shokhirev", "expertise": [ "Reviewer Expertise Bioinformatics" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWhile it is important to create a consistent and queryable database of biological identifiers, it is unclear what advances this tool brings to the field. For example, how does this tool compare to other queryable database tools such as mygene.info, or BioMart? The paper will greatly benefit from a comparison to these and other such tools.\nI downloaded and ran the tool but it seems I can't get through the update phase when I run ./biobtree update (It seems to hang after uniprot_reviewed finishes) without any other messages. When I rerun using biobtree --d uniprot_reviewed update it finishes but there is an error:\n\nError while reading file-> .//out/index/0_13.938476000.gz panic: gzip: invalid header\nI tried running generate and web after that regardless, but couldn't get it to work:\npanic: mdb_txn_commit: MDB_BAD_TXN: Transaction must abort, has a child, or is invalid\nError while reading meta information file which should be produced with generate command. Please make sure you did previous steps correctly.\nThe author needs to debug/test their code to ensure that it can be used by others.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No", "responses": [ { "c_id": "4474", "date": "10 Mar 2019", "name": "Tamer Gur", "role": "Author Response", "response": "Thank you for reviewing the article. I agree that there are several similar tools exist with different dataset and functionalities such as Biomart and mygene.info. However, this tool can still complement them for following main reasons. Biobtree can work in local machine. This can be especially useful when large number of requests needs to be performed. For instance currently similar Uniprot tool documentation suggests either split the requests or download underlying data when number of requests are above 50K. These types of limitations for bulk requests are sensible for fair usage of a public service and can be more suitable with Biobtree type locally runnable tool. Users custom dataset can be integrated. Tool provides new intuitive web interface. In relation to reported errors, I have added demo of tool in case such errors happen again. I have also added integration test which runs periodically on Linux, MacOS and Windows operating systems via Azure DevOps platform. These tests can be accessed publicly and test and demo links can be found at github page. Based on these tests, it seems that tool is working as expected. I believe that hanged process is a specific issue or bug which I am happy to resolve if I have more information. The rest of problems which have been reported are most probably due to the prematurely exited hanged process. Either starting in a new folder or passing --clean parameter can solve the issue." } ] }, { "id": "46335", "date": "15 Apr 2019", "name": "Samuel Lampa", "expertise": [ "Reviewer Expertise Scientific workflow tools", "Cheminformatics", "Semantic web." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes a commandline tool, Biobtree, that is claimed to allow to process relations between bioinformatics datasets based on various characteristics such as identifiers and keywords.\nThe manuscript describes the tool in a clear way technically, making it quite clear what it does in technical terms, and how it is supposed to be used.\nAlso, I was able to install and run the tool in a simple way on my laptop (i5 CPU, 8GB RAM and 10-15 GB free hard drive, Xubuntu 16.04 64 bit) without problems. It provides a simple but good looking and easy to use web interface.\nI'm seeing at least two major issues with the tool and manuscript though, that needs being thoroughly addressed to make them acceptable.\nMain problem 1: Visualization?\nFirstly, the title claims that the tool does visualization of the database produced by the tool. Perhaps I'm missing something, but I have not found any visualization in the tool apart from a form of search hit result listings. I don't think this is enough to be called \"visualization\". Especially as it is unclear how the current form of output is supposed to be used in a concrete biological usecase. With the current wording, I would expect something more graphical, like a graphviz-like graph view of dataset relations.\nSuggested edits to make the tool and paper acceptable:\nProvide graphical visualization beyond results listings (or explain how to show them, if I have missed them), or else remove \"visualization\" from the title and other places. Use this/these visualizations in the use cases/demonstrators discussed above, to explain how they contribute to solving concrete biological problems.\nMain problem 2: Lack of context and discussion of biological relevance\nThe first and main problem with the manuscript is that it does not provide a clear enough description of what biological problem it is solving. Nor does it provide an overview of existing tools and solutions in this field. Right now, the manuscript only states what the tool can do in technical terms. It somehow reads like a (well written) user guide or README file, but not yet a scientific paper. To help potential new users understand why they might need this tool, it needs to be put in context and compared with other existing tools.\nIn my view, the manuscript needs the following points thoroughly addressing to be acceptable:\nIn the introduction: Elaborate on the field of mapping/visualising dataset relations, mentioning relevant existing similar tools, what are the typical problems, and what particular problem Biobtree solves. Explain a few examples of biological problems that can be solved with this tool, or type of tool. E.g. in the results: Provide at least one, and optimally two or three potentially simple, but relevant, biological demonstrators or use cases, that can be addressed with the tool. Provide complete instructions on how to re-run this or these demo(s) and provide outputs for this/these in terms of figures or diagrams and how these were produced. In this way, both reviewers and users can make sure that they understand how to operate the tool. In the discussion: Connect back to the explained problem the tool is addressing, and explain how the problem was solved, again reinstating the relevance of this specific tool compared to other existing tools, and what improvement it provides to the end user trying to solve biological problems, exemplified by the demonstrators or use cases.\nLanguage issues\nThe manuscript also contains quite a number of language issues. I'm listing a few language suggestions below as examples, but further language proofing or editing is highly recommended, to make sure there are not more of these:\nMethods section: \"in GO programming language\" --> \"in the Go programming language\" (Note the \"the\" and that only G is uppercase in \"Go\"). Update phase section: \"to LMDB\" -> \"to the LMDB\" Update phase section: \"Updating reads selected datasets as a stream\" I don't understand this sentence. Please language-check it. \"is used in next\" -> \"is used in the next\" Generate phase section: \"the project github page\" -> \"the project's GitHub page\" Web phase section: \"starts web phase\" -> \"starts the web phase\" Web interface section: \"The Web interface allow user\" -> \"The web interface allows the user\" Operation section: \"Biobtree executable\" -> \"The biobtree executable\".\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-145
https://f1000research.com/articles/9-29/v1	20 Jan 20	{ "type": "Brief Report", "title": "Serum levels of tau protein increase according to the severity of the injury in DAI rat model", "authors": [ "Keisuke Tomita", "Taka-aki Nakada", "Taku Oshima", "Rui Kawaguchi", "Shigeto Oda", "Keisuke Tomita", "Taku Oshima", "Rui Kawaguchi", "Shigeto Oda" ], "abstract": "Traumatic brain injury (TBI) in the form of diffuse axonal injury (DAI) is difficult to diagnose in the early phase of the injury. Early diagnosis of DAI may provide opportunity for developing treatment and management strategies. Tau protein has been demonstrated to increase in the early phase of TBI with high diagnostic accuracy in patients with DAI. We tested the biological plausibility of tau protein using a rat DAI model by evaluating the association between serum tau levels and the severity of brain injury. DAI was induced in animals using the Marmarou model. After a survival of 60 minutes, rats were anesthetized and sacrificed after obtaining blood samples (5ml) from the heart. Eighteen rats were employed in the present study and were randomly subjected to sham-operated control (n=4), mild DAI (n=7), and severe DAI (n=7). Of seven severe DAI rats, two rats that had focal injury caused by skull fracture were excluded in the measurement of tau protein level. The serum levels of tau protein in the rat DAI model were found to increase significantly and consistently according to the severity of the injury. Rats with DAI showed significantly higher serum levels of tau protein compared to sham rats; the severe DAI rats had higher levels of tau than moderate DAI and sham rats (sham vs. mild, P=0.02; mild vs. severe, P=0.02). In conclusion, serum tau protein levels may be useful as a biomarker for diagnosing and estimating the severity of DAI in the early phase.", "keywords": [ "serum tau protein", "diffuse axonal injury", "traumatic brain injury", "biomarker" ], "content": "Introduction\n\nTraumatic brain injury (TBI) in the form of diffuse axonal injury (DAI) is difficult to diagnose with computed tomography (CT) scans, due to the minimal effect on the anatomical structure of the brain in the early phase of the injury. However, DAI is frequently associated with poor clinical outcome, due to the extensive shear disruption of the axons by rotational or acceleration forces of the head1,2.\n\nEarly diagnosis of DAI may contribute to developing treatment and management methods. Apart from imaging diagnostic techniques, biomarkers may be useful in diagnosing DAI and predicting its severity. We focused on tau, a protein which composes important structural elements in the axonal cytoskeleton3. Tau protein has been demonstrated to increase in the early phase of TBI with high diagnostic accuracy in patients with DAI4. In addition, blood tau protein level has been reported to increase in concussion and chronic traumatic encephalopathy5,6, and it has also been reported that there is an association with severity. However, the same effect has not been confirmed in DAI. Therefore, we tested the biological plausibility of tau protein using a rat DAI model by evaluating the association between serum tau levels and the severity of brain injury.\n\n\nMethods\n\nThe animal study protocol and procedures were reviewed and approved by the Animal Research Committee of Chiba University (approval number 26-341) following the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan). All procedures used in this study were optimized to minimize animal suffering and distress by carefully following the procedures and monitoring the effectiveness of the analgesics.\n\nMale Sprague-Dawley rats (13–14 weeks old, weighing 330–380g) were obtained from CLEA Japan, Inc. A total of eighteen rats were used for this study and were allocated to experimental groups using simple randomization. Each rat was housed one per cage; (270 × 440 × 187 mm; KN-601, Natsume Seisakusyo Co., Ltd., Japan) and standard corn cob cage bedding was used. The rats were kept under 12 h light and dark conditions with water and food ad libitum in the animal room at Chiba University, Japan. The diet consisted of standard laboratory feed (CLEA Rodent Diet CA-1, CLEA Japan, Inc.). Temperature (20-24°C) and humidity (50–55%) were also controlled. All rats were checked daily for general physical and health appearance, bedding/water bottle circumstances and any signs of distress.\n\nEach rat was selected randomly from the cage, and the procedures were performed one by one. DAI was induced in animals using the Marmarou model in the laboratory during light conditions7. The Marmarou model is recognized as one of the most commonly used to create DAI in rats. It is inexpensive, easy to perform and capable of producing graded DAI that closely mimics that seen in human TBI. Rats were anesthetized by intraperitoneal injections of medetomidine hydrochloride (0.375mg/kg), midazolam (2mg/kg) and butorphanol tartrate (2.5mg/kg)8. Then, the skull of the rat was exposed with a midline incision to adhere a helmet, a 10 mm diameter stainless steel disk with a thickness of 3mm, at the midline between the coronal and the lambdoid suture. Subsequently, rats were fixed on a foam bed in prone position. A weight (450g) impounder was allowed to fall freely through a Plexiglas guide tube from a predetermined height (severe group 2m; mild group 1m) to provide an impact to the helmet. After the impact, the stainless steel disk was removed and the incision was sutured. Sham-injured rats underwent the same surgical procedure but were not subjected to injury. Rats were housed and warmed at 37.0°C using a heating pad. After a survival time of 60 minutes, DAI and control rats were anesthetized by intraperitoneal injections of medetomidine hydrochloride (0.375mg/kg), midazolam (2mg/kg) and butorphanol tartrate (2.5mg/kg) and sacrificed by the method of cervical dislocation after obtaining blood samples (5ml) by puncturing the heart with a 23G needle. Serum was centrifuged at 1000 rpm for 20 min and stored at -80°C until analysis according to the instructions of enzyme linked-immunosorbent assay (ELISA) kit.\n\nSerum levels of tau protein were measured using a commercially available kit based on the principle of the sandwich ELISA (Cat. no. LS-F23602, LSBio, WA) according to the manufacturer’s instructions. Absorbance readings at wavelength 450nm were performed on the automated plate reader SpectraMax® M5e with SoftMax Pro Ver. 5.2. Measurements were performed in duplicates.\n\nIn the present study, serum tau protein levels of tau were compared between control and mild injury and between mild and severe injury using Mann-Whitney’s U-test. Since there was no data available to calculate the sample size from previous studies, two cases were first tested in each group. From these results, the estimated level of serum tau protein was 1300±200 pg/mL in the severe DAI group. Furthermore, we also estimated tau protein levels at 900 pg/mL in the mild DAI group and 500 pg/mL in the sham-injured group. In order to achieve the level of statistical significance of 0.05 with a power of 80%, data for four rats in each group were needed. A previous study had reported that death or skull fracture occurred in about 40% of the severe DAI group7. Therefore, we planned to use seven rats for the experiment in the severe DAI group. In addition, seven rats were used in the mild group in case of any adverse events occurring. All statistical analysis was performed with the GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA).\n\n\nResults\n\nEighteen rats were employed in the present study and were randomly subjected to sham-operated control (n=4), mild DAI (n=7), and severe DAI (n=7). The mean weight ± standard deviation (SD) and age ± SD in each group was 360.0±8.2g, 365.7±13.9g and 360.6±13.7g, and 94.3±2.6 postnatal days, 93.1±1.1 postnatal days and 93.2±1.3 postnatal days, respectively9. Of seven severe DAI rats, two rats that had focal injury caused by skull fracture were excluded in the measurement of tau protein level. The serum levels of tau protein in the rat DAI model were found to increase significantly and consistently according to the severity of the injury (Figure 1).\n\nSerum tau protein levels increased significantly as degree of diffuse axonal injury increased at one hour after injury (sham vs. mild, P=0.024, sham vs. severe, P=0.016, mild vs. severe, P=0.018). P values were calculated using Mann-Whitney’s U-test. Error bars indicate inter-quartile range. *P<0.05.\n\n\nDiscussion\n\nIn our study, the serum tau protein levels in the rat DAI model were found to increase significantly and consistently according to the severity of the injury.\n\nThe potential biomarkers of DAI such as S-100 calcium-binding protein B, neuron specific enolase, neuron filament, etc., have been evaluated for usefulness for diagnosis and prognostication, with limited success to date10–12. Tau protein is a microtubule-associated protein and has highly specific expression in neuronal axons3. In a previous animal study, serum tau levels were higher in rats subjected to focal brain injury compared to sham operated controls. The highest level was observed one hour after injury, compared to values at 6h, 24h, 48h, and 168h. The serum tau protein levels increased according to the severity of the injury (sham vs. mild, P<0.001; mild vs. severe, P<0.001), which was consistent with our results despite the difference in the injury model13. However, to the best of our knowledge, no study has demonstrated the relationship between the severity of DAI and serum tau protein level. By using the rat DAI model rat, we were able to demonstrate that the serum level of tau protein increases significantly and consistently according to the severity of DAI.\n\nThere are several limitations in this study. In Marmarou’s study, the severity of traumatic brain injury of rats was differentiated by the height of the weight drop and confirmed according to the mortality after impact. Although we followed the same procedures for producing the models in this study, we did not evaluate the severity of DAI. Furthermore, we only evaluated serum tau protein level at a single time point of one hour from traumatic brain injury. However, setting multiple timepoints may have contributed to a more precise evaluation of the relationship between serum tau protein level and severity of DAI.\n\nIn conclusion, the serum levels of tau protein may be a useful biomarker for diagnosing and estimating the severity of DAI in the early phase.\n\n\nData availability\n\nDRYAD: Serum levels of tau protein increase according to the severity of the injury in DAI rat model. https://doi.org/10.5061/dryad.3r2280gc99.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nGennarelli TA, Thibault LE, Adams JH, et al.: Diffuse axonal injury and traumatic coma in the primate. Ann Neurol. 1982; 12(6): 564–74. PubMed Abstract \| Publisher Full Text\n\nChelly H, Chaari A, Daoud E, et al.: Diffuse axonal injury in patients with head injuries: an epidemiologic and prognosis study of 124 cases. J Trauma. 2011; 71(4): 838–46. PubMed Abstract \| Publisher Full Text\n\nBuée L, Bussière T, Buée-Scherrer V, et al.: Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brain Res Brain Res Rev. 2000; 33(1): 95–130. PubMed Abstract \| Publisher Full Text\n\nTomita K, Nakada TA, Oshima T, et al.: Tau protein as a diagnostic marker for diffuse axonal injury. PLoS One. 2019; 14(3): e0214381. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShahim P, Tegner Y, Wilson DH, et al.: Blood biomarkers for brain injury in concussed professional ice hockey players. JAMA Neurol. 2014; 71(6): 684–92. PubMed Abstract \| Publisher Full Text\n\nRubenstein R, Chang B, Yue JK, et al.: Comparing Plasma Phospho Tau, Total Tau, and Phospho Tau-Total Tau Ratio as Acute and Chronic Traumatic Brain Injury Biomarkers. JAMA Neurol. 2017; 74(9): 1063–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarmarou A, Foda MA, van den Brink W, et al.: A new model of diffuse brain injury in rats. Part I: Pathophysiology and biomechanics. J Neurosurg. 1994; 80(2): 291–300. PubMed Abstract \| Publisher Full Text\n\nGaertner DJ, Hallman TM, Hankenson FC, et al.: Anesthesia and Analgesia for Laboratory Rodents. 2nd ed., Anesthesia and Analgesia in Laboratory Animals (London, UK: Academic Press, 2008). 239–97. Publisher Full Text\n\nTomita K, Nakada T, Oshima T, et al.: Serum levels of tau protein increase according to the severity of the injury in DAI rat model, v2. Dryad. Dataset. 2020. http://www.doi.org/10.5061/dryad.3r2280gc9\n\nChabok SY, Moghadam AD, Saneei Z, et al.: Neuron-specific enolase and S100BB as outcome predictors in severe diffuse axonal injury. J Trauma Acute Care Surg. 2012; 72(6): 1654–7. PubMed Abstract \| Publisher Full Text\n\nPelinka LE, Kroepfl A, Leixnering M, et al.: GFAP versus S100B in serum after traumatic brain injury: relationship to brain damage and outcome. J Neurotrauma. 2004; 21(11): 1553–61. PubMed Abstract \| Publisher Full Text\n\nZurek J, Bartlova L, Fedora M: Hyperphosphorylated neurofilament NF-H as a predictor of mortality after brain injury in children. Brain Inj. 2011; 25(2): 221–6. PubMed Abstract \| Publisher Full Text\n\nLiliang PC, Liang CL, Lu K, et al.: Relationship between injury severity and serum tau protein levels in traumatic brain injured rats. Resuscitation. 2010; 81(9): 1205–8. PubMed Abstract \| Publisher Full Text" }	[ { "id": "69132", "date": "17 Aug 2020", "name": "Vassilis E Koliatsos", "expertise": [ "Reviewer Expertise Traumatic brain injury", "neurodegenerative disease", "neuropathology", "axonopathy", "axons", "Wallerian degeneration", "brain-behavior correlations" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a simple straightforward report on the serum levels of tau protein, measured by ELISA, in two different settings of the impact-acceleration model of diffuse traumatic brain injury (presumably causing two degrees of severity of diffuse axonal injury [DAI]). The advantage of having such data available is to show that we may have (emphasis is on may) a peripheral reporter of the severity of diffuse TBI and, potentially, DAI, using a model that is very appropriate for the task.\nHowever, as the authors would be the first to acknowledge, this experiment uses only two degrees of severity of impact acceleration (IA) based on mechanical inputs without testing for severity of traumatic brain injury and certainly not severity of DAI by any of the available applicable measures, also without a time course for the observed changes that is essential for characterizing DAI. Therefore, the neuropathology of DAI has not been tested here and it would appear that the only part of the report where something like this could be mentioned is the Discussion—certainly not the title, where a more neutral term such as “severity of impact acceleration” would be more appropriate.\nAlso, with only two degrees of IA tested, the conditional “may” should be used throughout. We need more work before we can look at a definitive study linking peripheral tau protein levels to severity of diffuse traumatic brain injury and quite a bit more if we want to explore peripheral markers of DAI.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "74640", "date": "20 Nov 2020", "name": "Mieszko Olczak", "expertise": [ "Reviewer Expertise Central nervous system", "forensic medicine", "trauma" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study evaluates the association between serum tau levels and the severity of brain injury in an animal model.\nMethodology, statistical analysis, results, and discussion are done correctly. One of the drawbacks of the work is the small size of research groups. It should be assumed that the differences between animals within the research groups are minimal and the results are reproducible. I have no objections.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "74639", "date": "30 Nov 2020", "name": "Amanda Heslegrave", "expertise": [ "Reviewer Expertise Biomarkers of neurodegenerative disease" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study aims to investigate whether serum tau could be a useful biomarker for estimating the severity of diffuse axonal injury in the early stages after trauma. The study uses a method to induce injury which depends on a weight dropped from a height to provide impact onto a helmet fixed to the rats head; two different heights were used.\nThe design of the experiment is simple and also the outcome was one serum tau measurement. The experiment assumes that the rats in each group receive the same injury - which is not unreasonable but is there a way to check this? Also the threshold for injury could have been tested by using more height conditions.\nOverall the conclusions do not seem unreasonable; that tau is increased quickly in serum following neuronal injury.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-29
https://f1000research.com/articles/9-27/v1	17 Jan 20	{ "type": "Software Tool Article", "title": "Identifying orthologs with OMA: A primer", "authors": [ "Monique Zahn-Zabal", "Christophe Dessimoz", "Natasha M. Glover", "Monique Zahn-Zabal", "Christophe Dessimoz" ], "abstract": "The Orthologous Matrix (OMA) is a method and database that allows users to identify orthologs among many genomes. OMA provides three different types of orthologs: pairwise orthologs, OMA Groups and Hierarchical Orthologous Groups (HOGs). This Primer is organized in two parts. In the first part, we provide all the necessary background information to understand the concepts of orthology, how we infer them and the different subtypes of orthology in OMA, as well as what types of analyses they should be used for. In the second part, we describe protocols for using the OMA browser to find a specific gene and its various types of orthologs. By the end of the Primer, readers should be able to (i) understand homology and the different types of orthologs reported in OMA, (ii) understand the best type of orthologs to use for a particular analysis; (iii) find particular genes of interest in the OMA browser; and (iv) identify orthologs for a given gene. The data can be freely accessed from the OMA browser at https://omabrowser.org.", "keywords": [ "OMA", "Orthologous Matrix", "orthology", "phylogenetics", "Hierarchical orthologous groups", "Comparative genomics" ], "content": "Introduction\n\nEvolution is one of the fundamental principles of biology. A major concept in evolution is that of homology or the relationship between genes related by common ancestry. From this general homologous relationship, pairs of genes might be classified in any of various sub-groups of homologs, including ortholog, co-ortholog, paralog, in-paralog, out-paralog, xenolog, or homoeolog, among others.\n\nIn comparative genomics and phylogenetics, the fundamental concept of orthology relates “corresponding” genes in different species: orthologs are pairs of genes which have evolved from a single gene in the last common ancestor1. Among many applications (reviewed by Glover et al.2), orthologs are useful to infer species trees (reviewed by Fernandez et al.3) and tend to be functionally conserved (reviewed by Gabaldon and Koonin4). A wide range of methods have been developed to infer orthologs, including PANTHER, OrthoInspector, InParanoid, and OrthoDB, among others (reviewed by Altenhoff5). This primer focuses on OMA (Orthologous MAtrix), a widely used method and database for inferring orthologs between species, and described the OMA Browser6.\n\n\nMethods\n\nIn this section on understanding orthology, we focus specifically on i) the basic concepts of orthology, ii) defining the sub-types of orthology we infer in OMA, and iii) the key differences between the different types of orthologs, which can help to make a decision on what type to use for your own analyses.\n\nOrthology is a type of homologous relationship, specifically between pairs of genes in different species that originated by and started diverging due to a speciation event. Conversely, paralogy is a relationship between pairs of genes that started diverging due to gene duplication. Box 1 describes how to distinguish between terms referring to a relationship between genes and those referring to the genes themselves.\n\nHomology, orthology, paralogy, and any terms ending in “-gy” indicate a relationship between genes. Homolog, ortholog, and other “-log” terms denote the genes themselves. For example, if OMA infers a pairwise orthologous relationship between genes, these two genes are considered orthologs to each other.\n\nIn OMA, we infer and provide several sub-types of orthologs: pairwise orthologs, Hierarchical Orthologous Groups (HOGs), and OMA Groups. It is important to understand how these three categories of orthologs are different in order to choose the appropriate type for your analysis. The differences between the three sub-types of orthologs reported in OMA are all based on one main factor: how they are inferred. In the OMA algorithm, the pairwise orthologs are inferred first, and are then used to build the HOGs and OMA Groups.\n\nIn the following sections we go in depth into each type of ortholog individually. We use a toy example of an OMA run on human, mouse, and monkey genomes to illustrate the differences between the three types of orthologs we infer and provide in OMA.\n\nThe OMA algorithm7,8 starts by an all-against-all alignment to find homologs between all the genes in all the genomes (proteins and proteomes to be exact, as the algorithm uses amino acid sequences). The algorithm then proceeds to filter out out-paralogs, while still including in-paralogs (Figure 1). Considering any two genomes, a pair of genes that passes all the appropriate steps in the OMA pipeline will be considered pairwise orthologs. Currently, there are over 2000 species in the OMA database (accessed Oct 2019) which are used to perform a pairwise comparison of all genomes.\n\nThese pairwise orthologous relationships can be mapped and visualized on a graph. In our example, we consider three mammalian species: the human, monkey, and mouse (Figure 2A). Their genomes are shown in Figure 2B, with blue, red, and green circles representing the mouse, monkey, and human genes, respectively. In this example, there was a duplication of gene B that took place after the mammals’ speciation, giving rise to genes B1 and B2 in the monkey and human (Figure 2C). The genomes and the species tree are needed as input for the OMA pipeline.\n\nThe species tree and genomes are used as input for the OMA pipeline (A–B), which results initially in pairs of orthologs (D). The pairwise orthologs are then used to build orthologous groups (E), which are subsequently clustered into HOGs (F) or OMA Groups (G). Each grouping method results in slightly different pairs of orthologs (D, H, I).\n\nPairs of orthologs are inferred based on the sequence similarity of genes between genomes (Figure 1). This results in a list of pairwise orthologs (Figure 2D) which are subsequently used for building HOGs and OMA Groups. In OMA, we also report the relationship cardinality of the pairwise orthologs, which reflects the level of co-orthology, or the degree of duplications which one or both of the orthologs has undergone. One-to-one (1:1) pairwise orthology means that both genes in the pair have only one ortholog in the other species. A one-to-many relationship (1:m) means that the gene of interest has more than one ortholog in the other species. This implies that the gene was duplicated in an ancestor of the other species, but after the speciation event. A many-to-many (m:m) relationship means both orthologs underwent lineage-specific duplications.\n\nThe pairs of orthologs are then mapped to an orthology graph (Figure 2E), where each node on the graph represents a gene, and each solid line between the genes represents an inferred pairwise orthologous relationship. The graphs are then used as input to compute the HOGs. As one can imagine from the name, HOGs are a way to group, or cluster, pairs of orthologs. HOGs aim to identify sets of genes that have descended from a common ancestral gene in a given ancestral species (i.e. at a specific taxonomic level)9. Box 2 and Figure 3 explain the hierarchical nature of HOGs.\n\nThe “hierarchical” nature of HOGs is because they are defined with respect to specific taxonomic clades. Groups defined at more recent clades are encompassed within larger groups that are defined at older clades, thus making them nested subfamilies.\n\n(A) A hypothetical gene tree between a gene family in three different species. Each node on this reconciled gene tree is either a speciation node or a duplication node (star). HOGs are formed at each taxonomic level, two HOGs at the mammalian level, and one at the tetrapoda level. (B) HOG-derived ortholog pairs at each taxonomic level. At the tetrapod level, the ortholog pairs are more numerous. One common pitfall may be to incorrectly infer an orthologous pair at the tetropoda level between the blue dog gene and the red human gene. However, these genes can be traced back to a duplication event rather than a speciation event, and thus are paralogs. The HOG-derived orthologs at the mammalian level are more fine-grained because the duplication event has already taken place.\n\nHOGs are constructed by identifying groups in the graph of pairwise orthologs. Each of the connected components in the graph are putative gene families, comprised of genes which descended from a common ancestral gene. However, due to errors in the pairwise orthologs inference, some relations between genes are either spurious or missing. An example of a spurious pairwise relation is between the monkey A gene and the mouse B gene (Figure 2E). As part of the OMA algorithm, these links are cut.\n\nAfter building the orthology graph and removing erroneous pairwise orthologous relations, the HOGs are built (Figure 2F). This is done by grouping all the connected components in the orthology graph at each taxonomic level. Thus, in the example, at the mammalian level there are two HOGs, one for gene family A and one for gene family B. These two HOGs mean that there was an ancestral gene A and an ancestral gene B in the mammals’ common ancestor. However, at the primate level, there are three HOGs: A, B1, and B2 because there was a duplication of gene B in the primate ancestor.\n\nAfter forming the groups of HOGs, HOG-derived orthologs can be considered as any pairs of genes between species which are contained in the same HOG, given that the HOG is defined at the level of their last common ancestor. Oftentimes, after grouping connected components to make the HOGs, there might be some newly inferred pairwise orthologous relations that weren’t initially inferred earlier in the pipeline. In Figure 2, the pairwise relationship that was not initially inferred by the OMA algorithm can be inferred because it connects genes grouped together in the same HOG. Thus, in the list of HOG-derived orthologs (Figure 2H), we added the mouse B gene and the monkey B1 gene as orthologs. Box 3 explains a common misinterpretation concerning HOGs.\n\nConsidering all genes between two species in a HOG as orthologs is a misinterpretation of a HOG because HOGs are nested and hierarchical. Two genes from different species in a HOG may have arisen by duplication and would therefore be paralogs. Thus, all of the genes between two species which started diverging at the taxonomic level of the HOG are orthologs.\n\nFor more information on HOGs, refer to our YouTube tutorial.\n\nFinally, we infer OMA Groups. Box 4 explains that the term “OMA Groups” is specific to OMA. OMA Groups are cliques of orthologs based on the orthology graph. In mathematics, a clique is a part of a graph where each node in that part is connected to all other nodes in that same part. Thus, our example results in two OMA Groups, in which all the genes are connected to each other by pairwise orthologous relations (Figure 2G).\n\nWhile pairwise orthologs and hierarchical orthologous are commonly used terms, OMA Groups is a term specific to OMA.\n\nIn practice, however, the clique requirement is very stringent—a single missing edge can lead to a gene being excluded from a group. In the OMA algorithm, there is therefore a tolerance parameter, such that even if a subset of genes does not form a fully connected subgraph, as long as it is only missing a few genes, and as long as each gene in the subset belongs to a distinct genome, they can still form an OMA group. These almost fully connected subgraphs are sometimes referred to as “quasi-cliques”. However, for simplicity (and because the missing edges in a quasi-clique are believed to, in truth, exist), we will refer to those as cliques in the rest of this tutorial.\n\nIn terms of the orthology graph, both 1:1 orthologs and OMA groups form cliques, but OMA Groups form \"maximal cliques\". The OMA Groups by definition consist of strict orthologs, since every gene was initially inferred to be pairwise orthologs to every other gene. This high precision comes at the price of a relatively low recall10. Indeed, when looking at the OMA Group-derived pairs of orthologs in our example (Figure 2I), we can see that there are fewer pairs of genes compared to the initial set of pairwise orthologs and the HOG-derived set of pairwise orthologs. Box 5 explains a common misinterpretation concerning OMA Groups.\n\nOne common misconception is that OMA Groups are groups of 1:1 orthologs. This is not necessarily the case. If two genes are 1:1 orthologs, this implies that there are no co-orthologs of either gene. If co-orthologs are inferred, OMA Groups will only contain one of the co-orthologous copies. However, like sets of 1:1 orthologs, OMA Groups have the property that all members are orthologous to all other members of the same group.\n\nAs shown in the example in Figure 2, the specific algorithm which is used to infer and/or group the orthologs is what determines the subsequent differences between the pairwise orthologs, HOGs, and OMA Groups. The number of genomes and taxonomic levels considered ultimately influences the number of inferred orthologs, as well as the potential errors for each type.\n\nThe number of genomes used for the inference of the different ortholog types can help with the accuracy of the predictions. In principle, we can hope that the more genomes used for inference, the more robust the predictions. For the pairwise orthologs, a maximum of three genomes at a time is used for inference in the OMA algorithm: the all-against-all genome comparison step uses two genomes, plus a third genome for the witness of non-orthology step (Figure 1). However, there may be errors due to only comparing a small number of genomes. For example, many draft genome assemblies might have missing genes or fragmented genes which do not pass the length tolerance threshold, and this would cause the pairwise ortholog inference to miss pairs. Additionally, pairwise orthologs might be missed if they have a large evolutionary distance, such as highly divergent proteins like those between bacteria and humans.\n\nFor the HOGs, all genomes are used in the inference process10, increasing the information content. Since we infer HOGs starting at the bottom (leaves) of the species tree and go up to the root, we pass through every internal node. These internal nodes represent ancestral genomes at different taxonomic levels. Thus, even if there are some mistakes in the extant genomes, by comparing multiple species and building the HOGs from the bottom up, the better quality extant genomes help to infer more robust ancestral genomes, and thus more accurate ortholog predictions. Additionally, by comparing multiple species it is easier to see when an erroneous pairwise relationship needs to be cut.\n\nDue to the differences in grouping algorithms, we can see a difference in the number of pairwise, HOG-derived, and OMA Group-derived orthologs. Firstly, using the HOGs, there might be more orthologous pairs inferred simply on the basis of being clustered together in the same group at the same taxonomic level, whereas initially they were not inferred to be pairwise orthologs. Biologically-speaking, this may happen if some pairs have a large evolutionary distance and they may be on the threshold of what we consider to be an ortholog in OMA. Bear in mind that HOGs contain paralogs (Box 6). OMA Groups are also computed by taking all genomes into account, and like the HOGs, are based on the orthology graph. As discussed above, the more genomes used to infer orthologs, the more robust the predictions. OMA Groups are likely to have few mistakes, but have a low recall (won’t include all species, misses some orthologs). Thus, OMA Group-derived orthologs usually have the fewest number of genes. Furthermore, OMA Groups only contain a maximum of one representative gene per species; if multiple co-orthologs exist, OMA will choose one to be in the OMA Group (Box 4).\n\nHOGs may, and often do, contain paralogs. Since HOGs are clustered groups of genes that descended from a common ancestral gene, a HOG might contain several genes from the same species, if that gene has undergone duplication after the speciation of interest. These genes from the same species would then be considered in-paralogs. For example, in Figure 2, the red genes B1 and B2 are paralogs because they originated from a duplication event (Figure 2C), but are also contained in the same HOG (Figure 2F). This represents a one-to-many relationship between the mouse (blue) gene and the monkey (red) genes, because B has undergone a duplication after the mammalian speciation.\n\nMost importantly, HOGs versus pairwise orthologs give us a different level of abstraction. HOGs make it much easier to think in terms of ancestral genes across evolution, whereas pairwise orthologs are often more intuitive when the focus is on one gene of interest in an extant species. This is because orthology prediction can be more fine-grained and informative when considering multiple taxonomic levels (Figure 3). Furthermore, as the HOGs are computed for each taxonomic level, it is up to the user to decide which level they want to consider, depending on their species of interest. This can vary from the root HOG (deepest common ancestor for all the species in OMA), or it can be any taxonomic level below that (younger).\n\nAs mentioned above, we sometimes don’t group HOGs together in the OMA pipeline due to insufficient orthologous links. Conversely, we might erroneously group HOGs together to make abnormally large HOGs. This could be due to some promiscuous domains which are shared between many gene families. Thus it is important to remember that all orthology relationships provided by OMA are inferences, and as such are subject to inference errors. Orthology inference is often a difficult problem— results cannot be expected to be error-free and should be interpreted accordingly. Table 1 summarizes the main differences between the algorithms.\n\nImplementation. OMA is available both as a standalone software11 and a database12, which contains precomputed orthology information for over 2000 species.\n\nOperation. OMA’s general workflow proceeds as described above (Figure 1 and Figure 2). To use the database version interactively, it’s possible to use any modern browser on a desktop, tablet, or mobile phone. To access the database programmatically, there is a REST API, as well as Python and R libraries13. Finally, data can also be downloaded in various formats. The standalone software works on mac or Linux, on a personal computer or a high-performance cluster. The Use Cases section describes how to access the OMA database via the browser and to obtain orthologs.\n\n\nUse cases\n\nThe next section provides step-by-step protocols for performing several types of analyses using the OMA browser6. Two tasks are dealt with: (i) Finding a gene in the OMA database, and (ii) finding different types of orthologs for a gene using the OMA browser, namely pairwise orthologs, OMA Groups, and HOGs.\n\nNote that all results obtained in this tutorial are based on the June 2019 release of the OMA database. OMA protein, OMA HOG, and OMA Group identifiers are not stable across releases, and orthologs are subject to slight changes based on additional or updated genomes. Here, we explain how to do the aforementioned tasks using the OMA browser.\n\nStart by navigating to the OMA browser at https://omabrowser.org/oma/home/.\n\nExample: UniProtKB S1000P_HUMAN gene. Each gene (also known as an entry) in OMA has an OMA identifier, consisting of the five-letter UniProtKB species code and a unique 5-digit number. For example, the human S100 calcium binding protein P gene’s OMA identifier is HUMAN22168. One can search for a gene by either an identifier, a protein sequence or a full-text search in the OMA browser:\n\n1. Search for an identifier. Search for a gene ID on the browser by typing/pasting into the search bar on the home page, or at the top bar of any page (Figure 4). Box 7 explains the autocomplete function of the search.\n\nIn OMA, a search with either the UniProtKB ID (S100P_HUMAN) or primary accession number (P25815), RefSeq (NP_005971) or EMBL accession (AAH06819) all retrieve the entry HUMAN22168. Note that OMA does not support searching by UniProtKB secondary accession numbers or Unigene IDs. Box 8 explains the pitfall of using OMA entry identifiers.\n\n2. Search for a protein sequence. Copy and paste the protein sequence into the search bar and choose Protein sequence in the dropdown menu. In this example, the sequence \"MTELETAMGMIIDVFSRYSGSEGSTQTLTKGELKVLMEKELPGFLQSGKDKDAVDKLLKDLDANGDAQVDFSEFIVFVAAITSACHKYFEKAGLK\" retrieves the same entry, HUMAN22168. Exact sequence matches to entries from other species are also shown. Spaces and line numbers in the sequence will be ignored. Searching by protein sequence is recommended in order to avoid any ambiguity. However, should the expected entry not be retrieved, it is possible to use the Approximate Sequence Search function.\n\n3. Search for a keyword using the full-text search. Select ‘Full-text search’ in the drop down menu, and type your keyword to search for. This can be alternative identifiers; for example, a search with the Ensembl gene (ENSG00000163993), transcript (ENST00000296370) or protein (ENSP00000296370) identifiers, PubMed identifiers (PMID:15632002) all return our original gene, HUMAN22168. Other text that may be in the description of the gene can be used as well. Quotes (\") can be used to search for an exact sequence of words. For example, a full-text search for “S100 calcium-binding protein P” also retrieves HUMAN22168.\n\n4. Get more information about your gene. After searching for your gene, you will be taken to the gene’s page, which provides some external information. You can also find this by clicking on the Information tab. The information for our example gene, which corresponds to the human protein S100 calcium binding protein P, is shown in Figure 5. The information page includes the OMA ID, description, organism, locus, other IDs and cross-reference, domain architectures, and Gene Ontology annotations.\n\nIf you are lucky, OMA will autocomplete and suggest genes for you. If there’s an exact match, it will automatically take you to the information page of the gene you searched for.\n\nOMA entry identifiers can change from one release to the next, particularly for human and model species which are regularly updated. It is thus recommended to search for the UniProt primary accession number as it is a stable identifier.\n\nShown are the General Information and IDs and Cross-references sections in the Information tab on the OMA browser. This tab also contains the Domain Architecture, Gene Ontology, Protein Sequence and Coding Sequence sections (not shown).\n\nExample: P53_RAT. After finding your gene in the OMA browser, the next step is retrieving the orthologs. As described in the section What types of orthologs do we provide, we discuss the three different “types” of orthologs which OMA computes. The entry Information page in OMA provides links to orthologs (Figure 6). In the following section, we describe how to find each of the different types of orthologs.\n\nTabs located under the heading link to orthologs. From left to right: ‘Orthologs’ links to pairwise orthologs and indicates the number of orthologs, ‘Hierarchical Orthologous Groups’ to HOGs, and ‘OMA Groups’ to OMA Groups.\n\nFinding pairwise orthologs\n\nThe rat p53 protein acts as a tumor suppressor in many tumor types; it induces growth arrest or apoptosis depending on the physiological circumstances and cell type.\n\nFirst, retrieve the entry by searching for the identifier “P53_RAT.” As can be seen from the Orthologs tab, there are currently 40 pairwise orthologs. This value may change with the inclusion of new species in OMA releases. Clicking on the Orthologs tab returns the list all pairwise orthologs (Figure 7).\n\nAll relationship cardinalities between the rat p53 and the pairwise orthologs are displayed. Let us consider an example for each of the different relationships:\n\n1. The mouse (Mus musculus) entry is listed as having a “1:1 ortholog” relation to the rat p53 gene. This indicates that the rat p53 gene only has one ortholog in mouse and this ortholog is the only one in mouse.\n\n2. The two Mexican tetra or blind cave fish (Astyanax mexicanus) entries are given as having a “1:n ortholog” relation to the rat p53 gene. This indicates that the rat p53 gene has more than one ortholog in Mexican tetra but that both orthologous genes in Mexican tetra fish have only one orthologous gene in rat. This implies that the p53 gene was duplicated in an ancestor of the Mexican tetra, but after the speciation event.\n\n3. The zebrafish (Danio rerio) entry is listed as having a “m:1 ortholog” relation to the rat p53 gene. This signifies that the rat p53 gene has only one ortholog in zebrafish, but this orthologous gene has more than one ortholog in rat. This implies that the p53 gene was duplicated in an ancestor of rat, but after the speciation event.\n\n4. The two three-spined stickleback (Gasterosteus aculeatus) entries are listed as having a “m:n ortholog” relation to the rat p53 gene. This signifies that rat p53 has more than one orthologous gene in three-spined stickleback and these orthologous genes have more than one ortholog in rat. This implies that the gene was duplicated at least twice: in the lineage of rat (the query species) and in the lineage of three-spined stickleback (the other species), yet all descend from a common ancestral gene in the last common ancestor of the two species.\n\n5. Finally, another rat entry (RATNO10594) is listed as being a “close paralog” of the rat p53 gene. This term is used to describe the paralogous sequence pairs that are co-orthologous to at least one other entry reported in the list of orthologs. Or in other words, they are in-paralogs with respect to the last common ancestor of all the species represented among the orthologs.\n\nInterestingly, the human p53 gene is not listed so the human gene is not a pairwise ortholog of the rat p53 gene. However, both the human and rat p53 genes are found in Hierarchical group HOG:0430403, indicating that they are related.\n\nFinding OMA groups\n\nAn OMA group contain sets of genes which are all orthologous to one another within the group. This implies that there is at most one entry from each species in a group.\n\nClicking on the ‘OMA Groups’ tab in the rat p53 gene entry returns the OMA group 866514 (Figure 8). Box 9 explains the pitfall of using OMA group identifiers. The fingerprint RCPHHQS corresponds to OMA group 866514. The fingerprint is a subsequence that is specific to a certain group in the current OMA release.\n\nThe OMA Group identifier is not a stable identifier; it is release specific. OMA group fingerprints should be used to track a group between different releases.\n\nOMA group RCPHHQS contains 14 entries from 14 different species. These entries have either a “1:1 ortholog”, “1:n ortholog” or “m:1” relation to the rat p53 gene. Note that the entries have a similar domain architecture display to that of the pairwise orthologs.\n\nFinding HOGs\n\nHierarchical groups contain genes that descended from a single common ancestral gene within a given taxonomic range. Clicking on the ‘Hierarchical Orthologous Groups’ tab in the rat p53 gene entry returns the Hierarchical group HOG:0430403 (Figure 9).\n\nBy clicking on the down arrow, we are taken to two sub-tabs: one to visualize the HOG with the graphical viewer and one to view the HOG in the table viewer. First, visualize with the graphical viewer. This displays the interactive HOG visualization, known as iHam14. In this representation of the HOG, the species tree is displayed in the left panel, and the right side shows all the extant genes in the HOG belonging to these species. By moving over internal nodes in the species tree, one can see all the inferred ancestral genes at that taxonomic level, delimited by vertical lines. Extant genes which descended from those common ancestral genes are boxes contained within these lines— these genes result from duplications. For more information see: YouTube video “iHam: interactive visualisation of hierarchical orthologous groups,” and 14.\n\nThis HOG contains 103 genes from 83 species, with the rat p53 gene shown in green, and p53 orthologous genes shown in grey. With the hierarchy open at the deepest common level (Chordata), we see a second rat p53 gene (close paralog), a single mouse (Mus musculus) p53 gene on a branch directly linked to the rat p53 gene (1:1 ortholog), the two Mexican tetra (Astyanax mexicanus) p53 genes (1:n ortholog), a single zebrafish (Danio rerio) p53 gene (m:1 ortholog) but no three-spined stickleback (Gasterosteus aculeatus) p53 genes (m:n ortholog). The fact that some genes are found to be pairwise orthologs, yet do not show up in the HOG is an example of how the GETHOGs clustering algorithm might separate inferred pairwise orthologs into separate HOGs.\n\nBy clicking on the second sub-tab in the Hierarchical Orthologous Groups tab, we are taken to the table view for this HOG. Here, we can find the same information as in the iHam view, except in list format. By clicking on a given taxonomic level, we can see all the genes contained within that HOG, at that level. Again, the entries have a similar domain architecture. It is possible here to download the genes, sequences, or multiple sequence alignment.\n\nIt is important to remember that two genes from the same HOG yet in different species may be paralogs rather than orthologs (Box 6). If you want only HOG-induced orthologs, it is necessary to consider only species that started diverging at the common ancestral taxonomic level. One can use the OMA REST API13 function “/api/protein/<id>/hog_derived_orthologs” to obtain induced pairwise orthologs from a HOG (see jupyter notebook example at https://github.com/DessimozLab/f1000_OmaPrimer)15.\n\n\nDiscussion\n\nOrthology inference plays a central role in a variety of applications, including gene function prediction, phylostratigraphy, genome evolution, and phylogenomics (Figure 10). The transfer of knowledge of the function from model species to human genes remains a major application. This application, the propagation of annotations, is central to UniProt and a subset of the electronic (IEA) annotations in GOA. Orthologs obtained using OMA have been employed for practically all of the applications (Table 2).\n\nReproduced from Glover et al.2, Advances and Applications in the Quest for Orthologs, Molecular Biology and Evolution, msz150.\n\nOMA provides three types of orthologs: pairwise orthologs, OMA Groups and HOGs. To give a tangible example of how they compare, the protein IDs for the different types of orthologs for the rat p53 gene were obtained using the methods described in the Use Cases section and analyzed (jupyter notebook; Figure 11)15. The OMA group (RCPHHQS) contains the fewest entries and, as expected, has only one entry per species. There are 40 inferred pairwise orthologs for the rat p53 gene. Recall that there are two Mexican tetra or blind cave fish (Astyanax mexicanus) entries, as well as two three-spined stickleback (Gasterosteus aculeatus) entries - there are thus pairwise orthologs from 39 species. HOG:0430403 contains the most entries, 102, from 83 species. The Venn diagram in Figure 11 indicates that only seven entries are common to the pairwise, OMA group and HOG orthologs for the rat p53 gene. The majority of the OMA group orthologs are common to all three types. The greatest overlap is seen between the pairwise orthologs and the HOG orthologs. The HOG orthologs group contains the most entries which are restricted to this type of ortholog, in agreement with it being the least strict type of ortholog. Thus, each type of ortholog can yield more or less orthologs, with varying degrees of accuracy. The type of ortholog to use (or the intersection of all orthologs) depends on the downstream application.\n\nRATNO03710 (P53_RAT) is found in both the root-level OMA HOG:0430403 and OMA Group RCPHHQS, but is obviously not listed in the pairwise orthologs. It has thus been excluded from the comparison.\n\nEach ortholog type is best suited for particular applications. Pairwise orthologs are useful when comparing two species or to identify orthologs for a specific gene. OMA Groups are particularly useful when identifying marker genes for phylogenetic reconstruction. Finally, HOGs generalize the concept of orthology to more than two species at a time. They are thus particularly suited for phylogenetic profiling and the study of the evolution of a gene of interest. We explain in more detail specific applications of orthology and which subtypes of orthologs from OMA we recommend be used in each case.\n\nOMA can be used for the prediction of gene function. Orthologs tend to retain their ancestral function, more than paralogs4. One can use OMA Groups for a high accuracy of functional prediction since all the genes are orthologous to each other, indicating a high degree of conservation. Alternatively, one can use HOGs at the most relevant taxonomic level to propagate function to the genes in the rest of the HOG. This will allow for a fine-grained approach, potentially being able to tell the difference between paralogs which may have sub- or neofunctionalized.\n\nWith the framework of HOGs, one can essentially track the evolutionary history of genes and gene families. This can be extended beyond protein function, but also allow for tracing the evolutionary history and conservation of the particular traits by mapping certain characteristics onto their genes. For example, Hosp et al.17 analyzed the evolutionary conservation of mitochondrial protein acetylation in plant and animal species by using HOGs.\n\nAdditionally, one can also reconstruct ancestral genomes using the framework of the HOGs. This is done by using the information from the extant genomes to infer what happened along the branches of the tree, and parsimoniously inferring the ancestral genomes at the internal nodes. OMA does this implicitly with the GETHOGS algorithm9. Thus, for each gene family, we can reconstruct the evolutionary history in terms of gene duplication, retention, or loss of a given gene family. In OMA, the python library pyham is specifically designed for this purpose14.\n\nAnother example of an application of orthology is finding taxonomically restricted genes, which are those genes which are only present in one particular lineage of a species tree. Taxonomically restricted genes are biologically interesting because the functions of these genes may help explain the phenotypic and ecological peculiarities of the species or lineage. Pairwise orthologs may be sufficient if one is only searching for the genes specific for one species compared to another. However, an analysis on taxonomically-restricted genes would be more informative with multiple genomes, thus HOGs are recommended.\n\nPhylogenetic profiling, also referred to as phyletic profiling, is a way to elucidate gene function24. The basic principle is that gene families which share a similar pattern of gains or losses in different species over time are likely to be involved in the same network. Genes involved in the same network are often functionally related—i.e. involved in the same biological process. To perform a basic level of phylogenetic profiling, one could use the HOGs to obtain all the orthologs of a gene at a given taxonomic level. Based on the species tree, one would then annotate the gene in a given species as present or absent. For more information on how to do this with OMA, see 25.\n\nFurther applications related to phylogeny is phylogenomics, or the study of the entire repertoire of phylogenetic trees for all of the gene families. With this genome-scale data for multiple organisms, one can study the evolutionary relationships. One generally does phylogenomics by first making a multiple-sequence alignment of homologous genes, then inferring a gene tree. Thus, one can use the entire set of HOGs at a given taxonomic level to make trees.\n\nAdditionally, one can make a species tree with information from OMA. A species tree by definition relates taxonomic units that started diverging through speciation. Strict orthologs are good for this purpose, thus OMA Groups are recommended. For more info on how to build a phylogenetic species tree using orthologs from OMA, see the section “Phylogenetic Marker Gene Export” in 12.\n\nFinally, searching for candidate genes for genetic engineering is a practical application of using orthologs. For example, if one can only choose a certain number of candidate genes to screen experimentally, one could collect the orthologs in your crop from a model species. One could prioritize the genes for screening based on if they are at the intersection of orthology inference methods.\n\n\nConclusion\n\nOrthologs are important, with a wide variety of uses. OMA is a database and algorithm to provide orthologs, and we provide several sub-types of orthologs, namely pairwise orthologs, HOGs, and OMA Groups. All have their own properties due to the algorithm used to derive them. Therefore, each will be particularly well-suited for certain types of analyses. We hope this Primer will serve as a guide to help users of OMA to understand the different types orthologs, which analyses to use them for, and how to obtain them from the browser.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nSoftware availability\n\nOMA Browser available at: https://omabrowser.org/.\n\nSource code available from: https://github.com/DessimozLab/OmaStandalone/tree/v2.4.0.\n\nSource code for Jupyter Notebook used to generate ortholog comparison: https://github.com/DessimozLab/f1000_OmaPrimer.\n\nArchived source code of OmaStandAlone at time of publication: https://doi.org/10.5281/zenodo.35555956.\n\nArchived source code for Jupyter notebook at time of publication: https://doi.org/10.5281/zenodo.355530115.\n\nOMA Browser license: Mozilla Public License version 2.\n\nJupyter Notebook license: MIT License.\n\nThe underlying sequences and annotations may be subject to third-party constraints. Users of the data are solely responsible for establishing the nature of, and complying with, any such intellectual property restrictions.", "appendix": "Acknowledgments\n\nWe would like to thank Adrian Altenhoff for providing details of the OMA algorithm and implementing additional functions in the REST API. Additionally, Clement Train for parts of Figures. We also thank Kimberly Gilbert for her feedback on the manuscript.\n\n\nReferences\n\nFitch WM: Distinguishing homologous from analogous proteins. Syst Zool. 1970; 19(2): 99–113. PubMed Abstract \| Publisher Full Text\n\nGlover N, Dessimoz C, Ebersberger I, et al.: Advances and Applications in the Quest for Orthologs. Mol Biol Evol. 2019; 36(10): 2157–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFernández R, Gabaldón T, Dessimoz C: Orthology: definitions, inference, and impact on species phylogeny inference. arXiv [q-bio.PE]. 2019. Reference Source\n\nGabaldón T, Koonin EV: Functional and evolutionary implications of gene orthology. Nat Rev Genet. 2013; 14(5): 360–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltenhoff AM, Glover NM, Dessimoz C: Inferring Orthology and Paralogy. In: Anisimova M editor. Evolutionary Genomics: Statistical and Computational Methods. New York, NY Springer New York. 2019; 1910: 149–75. PubMed Abstract \| Publisher Full Text\n\nAltenhoff A, Dessimoz C, Alex WV, et al.: DessimozLab/OmaStandalone: V2.4.0 (Version v2.4.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3555595\n\nTrain CM, Glover NM, Gonnet GH, et al.: Orthologous Matrix (OMA) algorithm 2.0: more robust to asymmetric evolutionary rates and more scalable hierarchical orthologous group inference. Bioinformatics. 2017; 33(14): i75–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoth AC, Gonnet GH, Dessimoz C: Algorithm of OMA for large-scale orthology inference. BMC Bioinformatics. 2008; 9: 518. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltenhoff AM, Gil M, Gonnet GH, et al.: Inferring hierarchical orthologous groups from orthologous gene pairs. PLoS One. 2013; 8(1): e53786. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltenhoff AM, Boeckmann B, Capella-Gutierrez S, et al.: Standardized benchmarking in the quest for orthologs. Nat Methods. 2016; 13(5): 425–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltenhoff AM, Levy J, Zarowiecki M, et al.: OMA standalone: orthology inference among public and custom genomes and transcriptomes. Genome Res. 2019; 29(7): 1152–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltenhoff AM, Glover NM, Train CM, et al.: The OMA orthology database in 2018: retrieving evolutionary relationships among all domains of life through richer web and programmatic interfaces. Nucleic Acids Res. 2018; 46(D1): D477–85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaleb K, Vesztrocy AW, Altenhoff A, et al.: Expanding the Orthologous Matrix (OMA) programmatic interfaces: REST API and the OmaDB packages for R and Python [version 2; peer review: 2 approved]. F1000Res. 2019; 8: 42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrain CM, Pignatelli M, Altenhoff A, et al.: iHam and pyHam: visualizing and processing hierarchical orthologous groups. Bioinformatics. 2019; 35(14): 2504–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nnatashaglover: DessimozLab/f1000_OmaPrimer: First release (Version 1.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3555301\n\nSkunca N, Bošnjak M, Kriško A, et al.: Phyletic profiling with cliques of orthologs is enhanced by signatures of paralogy relationships. PLoS Comput Biol. 2013; 9(1): e1002852. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHosp F, Lassowskat I, Santoro V, et al.: Lysine acetylation in mitochondria: From inventory to function. Mitochondrion. 2017; 33: 58–71. PubMed Abstract \| Publisher Full Text\n\nTsagkogeorga G, Müller S, Dessimoz C, et al.: Comparative genomics reveals contraction in olfactory receptor genes in bats. Sci Rep. 2017; 7(1): 259. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchwager EE, Sharma PP, Clarke T, et al.: The house spider genome reveals an ancient whole-genome duplication during arachnid evolution. BMC Biol. 2017; 15(1): 62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShultz AJ, Sackton TB: Immune genes are hotspots of shared positive selection across birds and mammals. eLife. 2019; 8. pii: e41815. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSojo V, Dessimoz C, Pomiankowski A, et al.: Membrane Proteins Are Dramatically Less Conserved than Water-Soluble Proteins across the Tree of Life. Mol Biol Evol. 2016; 33(11): 2874–2884. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliams TA, Szöllősi GJ, Spang A, et al.: Integrative modeling of gene and genome evolution roots the archaeal tree of life. Proc Natl Acad Sci U S A. 2017; 114(23): E4602–E4611. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLopes KP, Campos-Laborie FJ, Vialle RA, et al.: Evolutionary hallmarks of the human proteome: chasing the age and coregulation of protein-coding genes. BMC Genomics. 2016; 17(Suppl 8): 725. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPellegrini M: Using phylogenetic profiles to predict functional relationships. Methods Mol Biol. 2012; 804: 167–77. PubMed Abstract \| Publisher Full Text\n\nMoi D, Kilchoer L, Aguilar PS, et al.: Scalable Phylogenetic Profiling using MinHash Uncovers Likely Eukaryotic Sexual Reproduction Genes. bioRxiv. 2019; [cited 2019 Nov 26]: 852491. Publisher Full Text" }	[ { "id": "58733", "date": "03 Feb 2020", "name": "Stephanie J. Spielman", "expertise": [ "Reviewer Expertise Molecular evolution and bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nZahn-Zabal et al. present a primer for ortholog identification in OMA, considering both a background of concepts and definitions and orthology as well as a practical guide for how to use the database. After revisions, this will be a very valuable resource for many biologists seeking to use this tool. The primary audience for this primer will be scientists using OMA for the first time, or similarly scientists seeking a more unified understanding of ortholog discovery. In order to clearly convey these concepts, the authors will need to substantially reorganize and edit the manuscript. Again, this paper has clear value, and I believe most necessary components are in the manuscript - there are some major organizational deficiencies. As such, although (nearly) all my comments are text-based, they should be regarded as \"major\" in terms of their impact on manuscript improvement. I believe that, given general guiding comments below, the authors will be able to update the manuscript largely at their discretion with some very careful editing:\nThe abstract of the manuscript presents a very nice list of objectives for readers, starting with \"(i) understanding homology and the different types of orthologs reported in OMA.\" This is a great first objective, but it is not achieved. In the opening paragraph of the Introduction, for example, the authors provide a list of types of orthologs, yet there are no clear definitions. A few paragraphs later in methods, the authors state the different kinds of orthologs OMA considers, but then each of these three types is considered entirely independently from each other with no unifying explanation until page 6. This will strongly hinder readers' ability to understand these subtypes, and it makes it very difficult for newcomers to know what THEY are looking for. Thus, the entire first half needs reorganization. I suggest this framework:\n\nWhat are homologs? What are orthologs?\n\nWhat terms are necessary to even understand OMA ortholog subtypes in the first place? What types of orthologs are in OMA, and what do they mean in relation to each other? Place Figure 2 much closer to intro will help, for example.\n\nFor each subtype, first define it, explain when one would want to find it and use it, and then how OMA obtains it.\n\nIn general, the authors need to go through and identify every term used (e.g. \"m:n ortholog\" and make sure it has a definition by the time of the term's introduction. It will probably be best to add a large-ish table showing all terms used close to the very beginning of the manuscript. Table 1 makes a good effort at this, except it does not appear until the \"Use cases\" section rather than the introduction/methods explaining what orthologs, which really detracts from the role Table 1 should play.\n\nI strongly encourage a careful read-through of _all the boxes_ to make sure they are appropriate for their location in the manuscript. Two examples:\n\nBox 1 uses the term \"pairwise orthologous relationship.\" This is not defined until the next page, in the last paragraph of \"pairwise orthologs.\" Readers cannot follow this. Moreover, and related to the main point in my #1 comment, pairwise orthologs themselves need to be defined in the very first sentence of the section describing them, rather than beginning with an algorithmic description.\n\nBoxes 8 and 9 discuss lack of ID stability. This concept was introduced in the main text 1-3 pages before, and neither box is referenced at that time.\n\nThe monkey/human/mouse example has some issues. First, \"monkey\" is not a species and should not be referred to as such. Second, it is sometimes written \"primate\" (see phylogeny image insert). Primate and monkey are not synonyms. Third, the authors need to adopt a color-blind friendly palette - they cannot assume red/green dots are distinguishable. Fourth, in almost every time these species (although, again, monkey is not a species, and the authors do need to clarify what animal is being considered) are listed in the main text, the list is in an entirely different order. More consistency is needed.\n\nIn the second section where the authors show some use-cases, the organization is much clearer. The discussion section is also overall fine. However, I was thrown a bit when the two first use cases considered entirely different genes. I would suggest the authors also use P53_RAT in \"Finding a gene,\" but this is not strictly necessary.\n\nIn general, for figures that are screenshots from OMA in the use cases, more description is needed in the form of a figure caption. There are currently just titles, no actual descriptions of the figures. As a primer, this manuscript should have more explanation for newcomers to interpret the figures in their entirety. Eg, what is the specific meaning of each box in Figure 9? And similar.\n\nThe provided links are fantastic and great to see, and they work! However, you may want to fix and/or hide the RuntimeWarning in the jupyter notebook, especially given the output about the user's full path.\n\nThe authors might want to change the title, if possible, to \"Identifying orthologs with Orthologous Matrix (OMA): A primer\".(or, vice versa with what goes into the parentheses). Just a thought, not necessary, and definitely at the authors' discretion.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? No\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "58732", "date": "10 Feb 2020", "name": "Paula Ramos-Silva", "expertise": [ "Reviewer Expertise comparative genomics", "proteomics", "transcriptomics", "biomineralization", "bacterial sporulation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThere are several public databases that provide orthologous groups, which rely on different approaches for orthology prediction. For most researchers is often difficult to keep up with all the algorithms and methods used in these databases in order to make good use of the available data.\nThis software tool article is guiding the reader through the usage of the OMA browser, a web-based interface for orthology prediction. The article is clear and well written. It provides an excellent guide for prospective users of the OMA browser.\n\nI have just a few comments that I believe will improve the first submitted version:\n\nGeneral comment:\n\nWhy should a researcher prefer the OMA browser for finding orthologs for his/her gene of interest instead of other platforms like KEGG (KO) or EggNOG etc? What are the biggest advantages of the OMA browser from the user perspective?\n\nSpecific comments:\n\nIn the Introduction:\n\nPage 3: The authors should clearly explain the differences between ortholog, co-ortholog, paralog, in/out-paralog, xenolog etc either by adding a new a box or a schematized figure.\n\nPage 3: “and described the OMA browser” – should be rephrased.\n\nPage 3: Figure 1 – how is the third genome selected?\n\nIn the Methods:\n\nPage 7: “OMA will choose one to be in the OMA Group (Box 4)”. Should be Box 5 instead of Box 4?\n\nPage 7: Table 1 – I suggest including a row with the candidate false positives and false negatives for each of the algorithms.\n\nPage 8: typo – S1000P_HUMAN. S100P_HUMAN is the correct ID.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-27
https://f1000research.com/articles/8-1007/v1	04 Jul 19	{ "type": "Research Article", "title": "Retrospective cohort study monitoring PEG-asparaginase activity in acute lymphoblastic leukemia patients with and without premedication", "authors": [ "Michael Losasso", "Bruce Bostrom", "Yoav Messinger", "Michael Losasso", "Yoav Messinger" ], "abstract": "Background: PEG-L-asparaginase (pegaspargase) is a critical component of therapy for children and adults with acute lymphoblastic leukemia (ALL). Allergic reactions, which may occur in up to one third of patients, are the major cause for discontinuation. One study reported lower rates of allergic reactions with premedication. Besides allergy, an unknown number of patients develop silent neutralizing antibodies not associated with allergic reactions. The purpose of this retrospective cohort study was to determine the incidence of silent inactivation of pegasparaginase and compare incidence of allergic reactions with and without premedication. Methods: Using a commercial assay, asparaginase activity was monitored following pegaspargase (2500 units/m2) in newly diagnosed children and young adults with B- and T-cell ALL from February 2013 to May 2017. The incidence of allergic reactions before and after initiation of premedication in May 2015 was compared. Results: One patient out of 59 (1.7%) had silent inactivation after the second dose. No patient had silent inactivation after the first pegaspargase dose and no standard risk B-cell ALL patients, who received only two pegaspargase doses in combination with oral dexamethasone, had silent inactivation. The incidence of grade 3 or 4 allergic reactions was 3.7% per dose with premedication (methylprednisolone, acetaminophen and diphenhydramine) versus 5.2% without. The incidence per patient with premedication given for most of the doses was 8.3% versus 17% without. These values are not statistically significant. Premedication did not affect pegaspargase activity. Conclusions: Due to the low incidence of silent inactivation with intravenous pegaspargase and the unlikely event patients receiving only two doses of pegasparaginase would receive erwinase for this possible transient silent inactivation, we recommend routine monitoring of pegaspargase activity only in patients scheduled to receive more than two doses.", "keywords": [ "Pegasparginase Activity", "Premedication", "Silent Inactivation", "Allergy", "Anaphylaxis" ], "content": "Introduction\n\nPEG-L-asparaginase (pegaspargase) is a critical component of therapy for children and adults with acute lymphoblastic leukemia (ALL). Its use is hampered by many issues including allergic reactions, silent inactivation, thrombosis, hyperbilirubinemia and pancreatitis1. Other common toxicities, such as hyperglycemia and hypertriglyceridemia, may be mitigated with the use of metformin and omega-3, respectively2,3. There is also ongoing interest in the use of carnitine to treat, and possibly prevent, hepatic toxicity, manifested by a severe increase in direct bilirubin, among other findings4.\n\nThe optimal dose, dose interval and target asparaginase level for pegaspargase is not completely established5. In pediatrics, a dose of 2500 units/m2 is the norm, whereas for adult patients, doses are often reduced due to increased toxicity at the pediatric dose. Some investigators have suggested using a pharmacokinetic driven model to individualize pegaspargase dosing6.\n\nThe use of premedication (acetaminophen, diphenhydramine and a corticosteroid) has been suggested as a possible means of reducing allergic reactions. In a multi-center study testing the use of pediatric-based regimens in young adults, the rate of grade 3 or 4 allergic reactions was reduced from 10% to 4% after premedication was mandated7. A study in adults with ALL reported allergic reactions in 7.2% of patients when pegaspargase was given concurrently with, or followed by, one week of prednisone8. Using a novel mouse model of asparaginase hypersensitivity, pretreatment with seven days of oral dexamethasone was the only agent capable of mitigating the severity of hypersensitivity and partially restoring asparaginase activity9. Dexamethasone given at the time of, or for one week following, asparaginase was not as effective.\n\nThe presence of antibodies against asparaginase may be found, from as early as the end of induction therapy10,11. The presence of asparaginase antibodies is a highly specific finding that is predictive of future allergic reactions but does not have the sensitivity to suggest modifications to therapy should be made10. The presence of asparaginase antibodies at end of induction did not appear to alter prognosis in a large multi-center study11. This suggests that measuring asparaginase activity is more useful than looking for the presence of antibodies.\n\nSilent inactivation of pegaspargase activity by anti-asparaginase antibodies or other immune-mediated mechanisms are potentially of greater concern than allergic reactions, as patients with allergic reactions to pegaspargase will be switched to erwinase, which theoretically will improve outcome. The true incidence of silent inactivation is unknown, as there are no reports of a comprehensive screening program for silent inactivation in a large multi-institutional trial. The largest published study found silent inactivation in 7/89 (8%) of patients12. However, these patients received induction with native Escherichia coli asparaginase before switching to pegaspargase, which is not current practice. The authors also report in the same group of patients that silent antibodies may spontaneously resolve with continued pegaspargase13. Prudence suggests that patients who receive premedications should have pegaspargase activity monitored after every dose, due to the possible but unproven concern that premedication will mask allergic reactions and silent inactivation. In fact, a consensus panel of experts recommends screening for silent inactivation in all patients undergoing therapy for ALL with asparaginase14.\n\n\nMethods\n\nAs the use of premedications and measurement of pegaspargase activity was considered by the leukemia provider group at Children’s Minnesota to be necessary for optimal care, no informed consent was obtained. Parents/adult patients were not informed of results unless intervention was indicated, which did not occur. This retrospective review study was approved by the institutional review board of Children’s Minnesota (IRB# 1606-062).\n\nThis retrospective study occurred in a large pediatric oncology center that diagnoses and treats approximately 40 new cases of ALL yearly in children and young adults up to age 30. If there are open studies, the patients are enrolled on Children’s Oncology Group protocols. Otherwise, patients are treated according to the most recent risk adapted protocols for standard risk B, high risk B and T-ALL. In order to reduce acquisition bias, charts of every patient who received pegasparaginase from December 2013 to September 2016 were abstracted (N=99). As this was a pilot study and the expected reduction of grade 3 or 4 allergic reactions with premedications was unknown at the time, sample sizes calculations could not be calculated. Data from all 99 patients were used to estimate the incidence of grade 3 or 4 allergic reactions by patient and by dose. For the detailed pharmacokinetic analysis, we used a subgroup of all patients from May 2014 to September 2016 (N=46) who had pegaspargase levels drawn. This number was sufficient to define the confidence intervals of the pegasparaginase activity.\n\nA total of 112 blood samples from these 46 patients were collected from a central venous portacath in conjunction with scheduled clinical visits from 3 to 12 days following pegaspargase administration at the standard dose of 2500 mg/m2. Pegaspargase was given by intramuscular injection or intravenously per Children’s Oncology Group protocols on an intermittent schedule starting with induction and completed prior to starting maintenance therapy. Because the distribution of the collection days clustered in ranges from day 3–5, 6–8 and 10–12, for analyses, pegaspargase activity was grouped in these categories. To better estimate the incidence of silent inactivation, pegaspargase levels lower than 0.01 units/ml were looked for in the data from an additional 13 patients, making a total of 59 evaluated. No evidence of silent inactivation was found in these 13 patients.\n\nThese patients were all treated according to Children’s Oncology Group protocols, using either intramuscular or intravenous pegaspargase as the only form of asparaginase. Intramuscular asparaginase was the standard of care until 2010 when intravenous administration became the new standard of care based on the Children’s Oncology Group AALL0932 protocol15. A comprehensive review of published studies concluded that the risk of grade 3 or 4 allergic reactions is independent of the pegaspargase route of administration15.\n\nWe became aware of an abstract showing a decrease in grade 3 or 4 allergic reactions in a multi-institutional study employing pegaspargase in young adults with ALL16. This prompted us to institute in May 2015 premedication with acetaminophen (10–15 mg/kg orally), diphenhydramine (1 mg/kg orally or intravenously), and methylprednisolone (1 mg/kg intravenously), within the hour prior to administering pegasparaginase. Every subsequent patient was to receive with all three of the premedication drugs without exception. The numbers with and without premedication are listed in Table 2 (per pegaspargase dose) and Table 3 (per patient).\n\nAllergic reactions to were graded per CTC 4.0 toxicity scales. We compared the incidence of grade 3 or 4 allergic reaction in patients with and without premedication, both per pegaspargase dose and per patient.\n\nRoutine monitoring of pegaspargase activity in patients with ALL was initiated in 2013 after the ‘asparaginase activity analysis’ test approved by Clinical Laboratory Improvement Amendments was introduced by AIBioTech, Richmond, VA 23225 US. Subsequent to the introduction in 2015 of a quantitatively identical test by Next Molecular Analytics, Chester, VA, samples were exclusively sent there.\n\nSPSS version 23 was used for graphing and analyses. Grouped data were displayed with box graphs depicting the 1st, 25th, 50th (median), 75th and 99th percentiles. The comparison of pegaspargase activity with and without premedication was done by independent sample t-test. The comparison of grade 3 or 4 allergic reactions by patient and pegaspargase dose with and without the use of premedication was done by Chi-squared analysis. As some premedication doses were missed due to omission by the treating physician, an additional analysis of the incidences of those who received premedication after every dose or most doses were compared to those who received no premedication before any dose. Missed pegaspargase activity samples were omitted from analysis (Table I).\n\n\nResults\n\nPharmacokinetic analyses were done on 112 specimens from 46 patients17. The 46 patients included 12 standard risk B-cell patients, 21 high risk B-cell ALL patients, and 13 T-cell ALL patients. There were 25 males and 21 females. The ages ranged from one to 29 years with a median of 8.3 years. The number of specimens and missed specimens per pegaspargase dose number are shown in Table 1. First dose specimens were frequently missed, whereas specimens on doses two to seven were collected at least half the time.\n\nFigure 1 is a box and whisker graph of pegaspargase activity on days 3–5, 6–8 and 10–12. The mean, standard error of the mean and standard deviation are: 1.37, 0.21 and 0.76 units/mL, respectively, for day 3–5; 0.89, 0.05 and 0.42 units/mL for day 6–8; and 0.89, 0.06 and 0.28 units/mL for day 10–12. These values are similar to those previously reported in pediatric patients with ALL18,19.\n\nData points outside of the whiskers of the 1st and 99th percentiles are represented by a circle (outlier more than 1.5 times the interquartile range) or star (extreme outlier more than three times the interquartile range). The attached number is a data point and not a value.\n\nFigure 2 is box and whisker graph of the pegaspargase activity on day 6–8, following doses with or without premedication. This time ranged was used for the comparison as it is the most common time for checking asparaginase activity. The mean and standard deviation for the no premedication group is 0.79 and 0.34 units/mL (N=14 samples), respectively, and for the premedication group is 0.92 and 0.41 units/mL (N=52 samples). These were not found to be significantly different by the independent sample t-test.\n\nThere was no significant difference in the activity with or without of premedication. Data points outside of the whiskers of the 1st and 99th percentiles are represented by a circle (outlier more than 1.5 times the interquartile range) or star (extreme outlier more than 3 times the interquartile range). The attached number is a data point and not a value.\n\nOnly one patient had silent inactivation with the following activity levels by dose number and day following pegaspargase activity was checked: dose 1, day 24 - 0.11 units/mL; dose 2, day 8 - 0.05 units/mL; dose 3 day 6 - 0.01 units/mL; dose 4 day 8 - 0.33 units/mL; dose 5, day 8 - 0.82 units/mL; and dose 6, day 10 - 0.62 units/mL. The low values after doses 2 and 3 were not reviewed due to a clerical error until after dose 4, which showed adequate activity, so pegaspargase was continued until the end of treatment.\n\nFor the analysis of the role of premedication in preventing grade 3-4 allergic reactions, the data was analyzed per pegaspargase dose and per patient. In the analysis per pegaspargase dose, premedication did not significantly reduce grade 3-4 allergic reactions. With premedication, 7/185 (3.7%) had grade 3-4 allergic reactions compared to 8/155 (5.2%) without premedication, p=0.5 (Table 2).\n\nTable 3 shows the incidence of grade 3-4 allergic reactions per patient. Without premedication, 7/42 (17%) had grade 3-4 allergic reactions. When premedication was given most of the time (usually the first dose was missed), 4/62 (6%) had grade 3-4 allergic reactions. When premedication was given for every dose, 4/32 (12%) had allergic reactions. There was no significant effect of premedication on grade 3-4 allergic reactions by dose when the premedication group (8/96; 8.3%) was compared to the no premedication group (7/42; 17%) (chi square = 2.09; p = 0.15) (Table 3). There was no difference in the distribution of patients who did or did not receive premedication by risk group.\n\n\nDiscussion\n\nCompared with historical controls that received similar therapy, premedication did not significantly reduce the incidence of grade 3 or 4 allergic reactions when measured per patient or per dose of pegaspargase. However, there was a downward trend in the incidence per patient when any use of premedication was compared to no premedication. As premedication does not negatively affect pegaspargase activity levels, and other studies using historical comparisons have suggested premedication may reduce allergic reactions, we are continuing the practice7,8.\n\nThe interesting observation by Tong et al. that asparaginase antibodies generated after native E. coli asparaginase may resolve while on pegaspargase continuation therapy needs to be confirmed in patients who receive only pegaspargase during induction and beyond15. We noted a transient decrease in pegaspargase activity, likely due to silent inactivating antibodies in 1/59 patients (1.7%). This decrease of pegaspargase activity occurred after the second dose in a high-risk B-cell ALL patient and resolved with continuation of pegaspargase dosing. No decrease in pegaspargase activity was seen in standard risk patients who received only two doses of pegaspargase in combination with oral dexamethasone.\n\nLimitations of the study include multiple missed activity levels that may have found addition patients with silent inactivation. The sample size also makes it difficult to estimate the true incidence of silent inactivation and if premedication reduces the incidence of grade 3 or 4 allergic reactions. Additional studies are needed to clarify this.\n\nContrary to the findings of a large multi-institutional trial, where the introduction of premedication significantly reduced the incidence of high-grade allergic reactions, our study did not show a statistically significant reduction with the use of premedication7. Despite this, we continue to use premedication in all patients receiving pegasparaginase. Due to the low incidence of silent inactivation we are only monitor asparaginase activity in patients who are planned to receive more than two doses of pegaspargase during their entire course of treatment (T-cell ALL and high-risk B-cell ALL).\n\nA recent publication by the pediatric oncology group at Johns Hopkins also reported significant reduction of infusion reactions and need for erwinase using premedication of H1 and H2 histamine antagonists. They also found a low incidence of silent inactivation with intravenous pegaspargase in one of 68 patients (1.5%), similar to our finding of one in 59 patients (1.7%)20.\n\n\nData availability\n\nFigshare: Data Set for Retrospective cohort study monitoring Pegaspargase activity in acute lymphoblastic leukemia patients with and without premedication Lossaso M, Messinger Y, Bostrom B. https://doi.org/10.6084/m9.figshare.8281826.v117\n\nThis project contains the following underlying data:\n\n- PEG Data De-identifyed.xlsx (demographic, medical and treatment information for each patient)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors would like to thank the Children’s Minnesota leukemia nurse case managers who did and continue to do an outstanding job to ensure PEG activity is collected on as many patients as possible.\n\n\nReferences\n\nStock W, Douer D, DeAngelo DJ, et al.: Prevention and management of asparaginase/pegasparaginase-associated toxicities in adults and older adolescents: recommendations of an expert panel. Leuk Lymphoma. 2011; 52(12): 2237–53. PubMed Abstract \| Publisher Full Text\n\nBostrom B, Uppal P, Chu J, et al.: Safety and efficacy of metformin for therapy-induced hyperglycemia in children with acute lymphoblastic leukemia. J Pediatr Hematol Oncol. 2013; 35(7): 504–8. PubMed Abstract \| Publisher Full Text\n\nBostrom B: Successful management of extreme hypertriglyceridemia from pegaspargase with omega-3. Pediatr Blood Cancer. 2012; 59(2): 350. PubMed Abstract \| Publisher Full Text\n\nAlshiekh-Nasany R, Douer D: L-Carnitine for Treatment of Pegasparaginase-Induced Hepatotoxicity. Acta Haematol. 2016; 135(4): 208–10. PubMed Abstract \| Publisher Full Text\n\nSalzer W, Bostrom B, Messinger Y, et al.: Asparaginase activity levels and monitoring in patients with acute lymphoblastic leukemia. Leuk Lymphoma. 2017; 18: 1–10. PubMed Abstract \| Publisher Full Text\n\nDouer D, Aldoss I, Lunning MA, et al.: Pharmacokinetics-based integration of multiple doses of intravenous pegaspargase in a pediatric regimen for adults with newly diagnosed acute lymphoblastic leukemia. J Clin Oncol. 2014; 32(9): 905–11. PubMed Abstract \| Publisher Full Text\n\nStock W, Luger SM, Advani AS, et al.: A pediatric regimen for older adolescents and young adults with acute lymphoblastic leukemia: results of CALGB 10403. Blood. 2019; 133(14): 1548–1559. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAldoss I, Douer D, Behrendt CE, et al.: Toxicity profile of repeated doses of PEG-asparaginase incorporated into a pediatric-type regimen for adult acute lymphoblastic leukemia. Eur J Haematol. 2016; 96(4): 375–80. PubMed Abstract \| Publisher Full Text\n\nFernandez CA, Smith C, Karol SE, et al.: Effect of premedications in a murine model of asparaginase hypersensitivity. J Pharmacol Exp Ther. 2015; 352(3): 541–551. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu C, Kawedia JD, Cheng C, et al.: Clinical utility and implications of asparaginase antibodies in acute lymphoblastic leukemia. Leukemia. 2012; 26(11): 2303–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKo RH, Jones TL, Radvinsky D, et al.: Allergic reactions and antiasparaginase antibodies in children with high-risk acute lymphoblastic leukemia: A children's oncology group report. Cancer. 2015; 121(23): 4205–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTong WH, Pieters R, Kaspers GJ, et al.: A prospective study on drug monitoring of PEGasparaginase and Erwinia asparaginase and asparaginase antibodies in pediatric acute lymphoblastic leukemia. Blood. 2014; 123(13): 2026–2033. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTong WH, Pieters R, Tissing WJ: Desensitization protocol should not be used in acute lymphoblastic leukemia patients with silent inactivation of PEGasparaginase. Haematologica. 2014; 99(7): e102–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan der Sluis IM, Vrooman LM, Pieters R, et al.: Consensus expert recommendations for identification and management of asparaginase hypersensitivity and silent inactivation. Haematologica. 2016; 101(3): 279–85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeaupin LK, Bostrom B, Barth MJ, et al.: Pegaspargase hypersensitivity reactions: intravenous infusion versus intramuscular injection - a review. Leuk Lymphoma. 2017; 58(4): 766–772. PubMed Abstract \| Publisher Full Text\n\nAdvani AS, Sanford B, Luger S, et al.: Frontline-Treatment Of Acute Lymphoblastic Leukemia (ALL) In Older Adolescents and Young Adults (AYA) Using a Pediatric Regimen Is Feasible: Toxicity Results of the Prospective US Intergroup Trial C10403 (Alliance). Blood. 2013; 122(21): 3903. Reference Source\n\nBostrom B: Data Set for Retrospective cohort study monitoring Pegaspargase activity in acute lymphoblastic leukemia patients with and without premedication Lossaso M, Messinge Y, Bostrom B. 2019. http://www.doi.org/10.6084/m9.figshare.8281826.v1\n\nAngiolillo AL, Schore RJ, Devidas M, et al.: Pharmacokinetic and pharmacodynamic properties of calaspargase pegol Escherichia coli L-asparaginase in the treatment of patients with acute lymphoblastic leukemia: results from Children's Oncology Group Study AALL07P4. J Clin Oncol. 2014; 32(34): 3874–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilverman LB, Supko JG, Stevenson KE, et al.: Intravenous PEG-asparaginase during remission induction in children and adolescents with newly diagnosed acute lymphoblastic leukemia. Blood. 2010; 115(7): 1351–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCooper SL, Young DJ, Bowen CJ, et al.: Universal premedication and therapeutic drug monitoring for asparaginase-based therapy prevents infusion-associated acute adverse events and drug substitutions. Pediatr Blood Cancer. 2019; 66(8): e27797. PubMed Abstract \| Publisher Full Text" }	[ { "id": "53512", "date": "26 Sep 2019", "name": "Bernard L. Marini", "expertise": [ "Reviewer Expertise Asparaginase", "leukemia treatment", "therapeutic drug monitoring" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDrs. Losasso, Bostrom, and Messinger should be applauded for their work in characterizing asparaginase activity levels in ALL patients. The results and research are clearly presented and accurately reported. The conclusions of the study are appropriate, and the limitations of this retrospective cohort study are well-described.\nSome specific suggestions/comments/recommendations:\nAbstract:\nI would include the n in the incidence of hypersensitivity reactions, or acknowledge that the study may not have been powered to assess this outcome. Readers who only look at the abstract may wrongly conclude pre-medication is not effective, when the analysis may have been underpowered to detect a statistically significant difference in hypersensitivity reactions in the pre-medication vs. no pre-medication group.\n\nThe paper, methods, and title are focused on the impact of pre-medication on activity levels; however, the conclusion primarily talks about the low incidence of silent inactivation with two doses; thus monitoring only being necessary in patients that receive >2 doses. I would consider a sentence re: pre-medication here as well.\n\nIntro:\nNot essential, but could consider mentioning fibrates in the treatment of hypertriglyceridemia, which is another first line option.\n\nWould cite more recent data on the impact of pre-medication on preventing hypersensitivity reactions - Cooper SL, Young DJ, Bowen CJ, Arwood NM, Poggi SG, Brown PA. Universal premedication and therapeutic drug monitoring for asparaginase-based therapy prevents infusion-associated acute adverse events and drug substitutions. Pediatr Blood Cancer. 2019 Aug;66(8):e27797.1 and Marini BL, Brown J, Benitez L, Walling E, Hutchinson RJ, Mody R, Jasty Rao R, Slagle L, Bishop L, Pettit K, Bixby DL, Burke PW, Perissinotti AJ. A single-center multidisciplinary approach to managing the global Erwinia asparaginase shortage. Leuk Lymphoma. 2019 May 17:1-152.\n\nI would be careful with stating asparaginase antibodies are always predictive of future allergic reactions, as not all antibodies inactivate asparaginase or lead to clinical hypersensitivity reactions (ex: CCG01961). Consider rewording to soften this language.\n\nIn paragraph 5, the statement is made that silent inactivation is of greater concern than allergic reactions. However, one could argue that allergic reactions are more common than silent inactivation with asparaginase (~1%), and monitoring activity levels in the setting of grade 1-2 allergic reactions is essential to determine which patients with clinical hypersensitivity reactions are having true antibody mediated reactions vs. other, non-antibody mediated (possibly ammonia vs. other immune-mediated) allergic reactions that would not warrant a switch to another asparaginase preparation.\n\nOther large scale studies assessing the rate of silent inactivation and finding the rate to be <8% could also be included in this discussion (or cited). These include, AALL07P4, Schore, et al. Leuk Lymph 2019 3, CCG1962, and Park JH, et al. Blood. 2016;128:16294.\nMethods:\nWhile a sample size was not calculated a priori, could the authors conduct a sensitivity analysis on the data determined the detectable effect size at 80% power in the primary outcome of interest (rate of grade 3/4 allergic reactions?)?\n\nWere dose reductions in PEG ever conducted in this data set for toxicity/age/risk factors for hepatotoxicity?\n\nIt is unclear how silent inactivation is defined in this data-set from the methods. It may also be clinically relevant to capture \"accelerated clearance\", which also may be clinically relevant. Not all anti-asparaginase antibodies lead to full inactivation of the drug, but some lead to accelerated clearance and lower than expected values at early time points. A patient with silent inactivation would have a peak level that is undetectable, whereas a patient with accelerated clearance may clear the PEG by day ~7 when an expected duration of asparagine depletion is 21-28 days with a full pediatric dose.\n\nWere any levels obtained in patients with low-grade hypersensitivity reactions? If so, did any of these patients have accelerated clearance/\"loud\" inactivation?\n\nWere the activity levels normally distributed? If not, perhaps medians in the box plot and a Mann Whitney U for comparison would be more accurate. Similarly, if the values in the 2x2 contingency table are small, consider a Fisher's exact test rather than chi-square for the incidence of hypersensitivity reaction comparison (although it likely won't make a significant difference).\n\nWas there a protocol to say every patient should have a level with every dose, or were levels obtained at random/provider discretion? Could there then be a selection bias in the patients who have activity levels obtained since so many were missed? I'm assuming first dose levels were \"missed\" because the risk of inactivation with the first dose should theoretically be zero, so they were perhaps more accurately intentionally omitted.\n\nResults:\n\nIs there any possibility that the level on day 6-8 of 2.8 was spurious? Were there any concomitant levels in this patient with this dose to make sense of such a high level?\n\nThe one patient being described as having silent inactivation - I do not think I would characterize this as silent inactivation technically, since they had detectable activity levels at several time points, and no peak level is ever undetectable. I would consider this a transient accelerated clearance with dose 2 and 3. If silent inactivation was truly present, a patient should not have activity present for a full week and then have full activity with later doses. Patients with activity levels even as low as ~0.02 may still have asparagine depletion (AALL07p4).\n\nIt may be interesting to graph levels over time in some of these patients with very low levels. Was there ever more than one level obtained per dose? Were repeat levels obtained to verify the low activity levels or follow the activity level trend over time (as is suggested in some guidelines?). This would help characterize the PK better in these patients.\n\nWas the choice to pre-medicate done at a strict, uniform point in time (i.e., was this a quasi-experimental design where a protocol to pre-medicate all patients was mandated?), or was pre-medication at the choice of the treating provider? If this was selected by the provider and not systematic, could there have been some slight selection bias where patients perceived to be at higher risk of hypersensitivity reactions were given pre-medication? This could have diminished the impact of pre-medication on reducing the rate of hypersensitivity reactions. Consider this in the discussion section if deemed relevant.\nDiscussion:\nI would more accurately call the silent inactivation here \"accelerated clearance\" as the patient had asparagine depletion for at least a week, even with the low level.\n\nConsider discussing the possible value of obtaining activity levels in patients with questionable allergic reactions (grade 1/2).\n\nCould add as a limitation that it appears patients only had one activity level obtained per dose, which makes it challenging to interpret PK, especially in patients with higher or lower than expected activity levels.\n\nOverall, a very well-done paper with logical conclusions. Thank you for allowing me to review the manuscript and for your hard work in this research.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5114", "date": "17 Jan 2020", "name": "Bruce Bostrom", "role": "Author Response", "response": "Bernard L. Marini, Department of Pharmacy Services and Clinical Sciences, Michigan Medicine, University of Michigan College of Pharmacy, Ann Arbor, MI, USA Drs. Losasso, Bostrom, and Messinger should be applauded for their work in characterizing asparaginase activity levels in ALL patients. The results and research are clearly presented and accurately reported. The conclusions of the study are appropriate, and the limitations of this retrospective cohort study are well-described.Some specific suggestions/comments/recommendations:Abstract: I would include the n in the incidence of hypersensitivity reactions, or acknowledge that the study may not have been powered to assess this outcome. Readers who only look at the abstract may wrongly conclude pre-medication is not effective, when the analysis may have been underpowered to detect a statistically significant difference in hypersensitivity reactions in the pre-medication vs. no pre-medication group.Response: done. Number of patients was added. Dr Marini is correct that the study was underpowered and we have added statements to that effect in the abstract and first paragraph of the discussion. The paper, methods, and title are focused on the impact of pre-medication on activity levels; however, the conclusion primarily talks about the low incidence of silent inactivation with two doses; thus monitoring only being necessary in patients that receive >2 doses. I would consider a sentence re: pre-medication here as well. Response: done. Sentence added: “Though premedication resulted in lower allergic reactions the difference was not statistically different due to small numbers.”Intro: Not essential but could consider mentioning fibrates in the treatment of hypertriglyceridemia, which is another first line option.Response: done and referenced. Would cite more recent data on the impact of pre-medication on preventing hypersensitivity reactions - Cooper SL, Young DJ, Bowen CJ, Arwood NM, Poggi SG, Brown PA. Universal premedication and therapeutic drug monitoring for asparaginase-based therapy prevents infusion-associated acute adverse events and drug substitutions. Pediatr Blood Cancer. 2019 Aug;66(8):e27797.1 and Marini BL, Brown J, Benitez L, Walling E, Hutchinson RJ, Mody R, Jasty Rao R, Slagle L, Bishop L, Pettit K, Bixby DL, Burke PW, Perissinotti AJ. A single-center multidisciplinary approach to managing the global Erwinia asparaginase shortage. Leuk Lymphoma. 2019 May 17:1-152. Response: Reference 1 is already there in the addendum and added the reference 2 to the addendum. I would be careful with stating asparaginase antibodies are always predictive of future allergic reactions, as not all antibodies inactivate asparaginase or lead to clinical hypersensitivity reactions (ex: CCG01961). Consider rewording to soften this language.Response we now quote exactly the sensitivity and specificity reported by the St. Jude group [Liu et al. Leukemia. 2012 Nov;26(11):2303-9] which addresses your concern. In paragraph 5, the statement is made that silent inactivation is of greater concern than allergic reactions. However, one could argue that allergic reactions are more common than silent inactivation with asparaginase (~1%), and monitoring activity levels in the setting of grade 1-2 allergic reactions is essential to determine which patients with clinical hypersensitivity reactions are having true antibody mediated reactions vs. other, non-antibody mediated (possibly ammonia vs. other immune-mediated) allergic reactions that would not warrant a switch to another asparaginase preparation. Response: we altered the paragraph somewhat and specified: “…potentially of greater concern than bone-fide allergic reactions, as patients with grade 3-4 allergic reactions to pegaspargase will be switched to erwinase, which theoretically will improve outcome.”As you suggested at the end of the paragraph we also added this sentence: “Additionally, the low grade 1-2 allergic reactions that are more common than silent inactivation do require pegaspargase activity monitoring to ensure a switch to erwinase if confirmed as true inactivation.” Other large scale studies assessing the rate of silent inactivation and finding the rate to be <8% could also be included in this discussion (or cited). These include, AALL07P4, Schore, et al. Leuk Lymph 2019 3, CCG1962, and Park JH, et al. Blood. 2016;128:16294.Response: Done. Also quoted Place et al. “Notably, lower silent inactivation with pegaspargase than native Escherichia coli asparaginase have been reported. [15-17]” Methods: While a sample size was not calculated a priori, could the authors conduct a sensitivity analysis on the data determined the detectable effect size at 80% power in the primary outcome of interest (rate of grade 3/4 allergic reactions?)?Response: A sentence in first paragraph of the discussion was changed as follows: “Compared with historical controls that received similar therapy, premedication did reduce the incidence of grade 3 or 4 allergic reactions when measured per patient or per dose of pegaspargase. The power calculation was only 0.54 per patient and 0.24 per pegasparaginase dose thus our study was underpowered to show statistical significance.” Were dose reductions in PEG ever conducted in this data set for toxicity/age/risk factors for hepatotoxicity? Response: No none. All PEG doses were given at a dose of 2500 units/m2, as documented in methods. It is unclear how silent inactivation is defined in this data-set from the methods. It may also be clinically relevant to capture \"accelerated clearance\", which also may be clinically relevant. Not all anti-asparaginase antibodies lead to full inactivation of the drug, but some lead to accelerated clearance and lower than expected values at early time points. A patient with silent inactivation would have a peak level that is undetectable, whereas a patient with accelerated clearance may clear the PEG by day ~7 when an expected duration of asparagine depletion is 21-28 days with a full pediatric dose. Response: we would suggest that prior studies defined silent inactivation similarly to our study. Here none of the measurements in the current study have included peak levels. Thus, like others we would suggest that accelerated clearance without noted clinical symptoms is the same as silent inactivation. Were any levels obtained in patients with low-grade hypersensitivity reactions? If so, did any of these patients have accelerated clearance/\"loud\" inactivation?Response: As noted – we did not collect low-grade (level 1-2) reactions therefore we cannot answer this question unfortunately. Were the activity levels normally distributed? If not, perhaps medians in the box plot and a Mann Whitney U for comparison would be more accurate. Similarly, if the values in the 2x2 contingency table are small, consider a Fisher's exact test rather than chi-square for the incidence of hypersensitivity reaction comparison (although it likely won't make a significant difference).Response: The data was normally distributed by visual inspection of the normal curve and Kolmogorov-Smirnov test of normality, WITH THE EXCEPTION of the day 6-8 pegasparaginase activity level. As Dr Marini pointed out this contained an extreme outlier which we could not explain. Thus we elected to remove this outlier and the distribution was normal. The removal of this was discussed in the methods.Data was reanalyzed and graphs redone after this change. Analysis showed a statistically significant difference in the day 6-8 pegaspargase level with and without premeds (lower without premed. Of note a significant difference was also seen with the dataset containing the outlier. Also analysis of the day 3-5 and 10-12 pegaspargase levels did not show a significant difference with premeds perhaps due to the small sample sizes.The finding of difference in the pegasparaginase level with premeds has not been previously described to our knowledge. This may be of clinical significance and should, if possible confirmed in other retrospective studies. Given the increased acceptance of premeds for PEG it is unlikely and perhaps unethical to confirm in a prospective randomized trial, in our opinion. Text relating to this new finding has been added to the manuscript in various places. Was there a protocol to say every patient should have a level with every dose, or were levels obtained at random/provider discretion? Could there then be a selection bias in the patients who have activity levels obtained since so many were missed? I'm assuming first dose levels were \"missed\" because the risk of inactivation with the first dose should theoretically be zero, so they were perhaps more accurately intentionally omitted. Response: our protocol did instruct that all pegaspargase doses will be followed by levels, as outlined in the methods. Compliance with this instruction was not complete at all as we outlined in the results. We think that there was no collection bias but rather non-compliance. It is true also that there is a misconception that risk of inactivation after the first dose is zero. Please note the recent report from Mary Relling group suggesting that many allergic reactions are actually to the PEG and may occur after the 1st dose of pegaspargase. We in fact have seen patients develop anaphylaxis right after the 1st dose. (Liu Y, Smith CA, Panetta JC, Yang W, Thompson LE, Counts JP, Molinelli AR, Pei D, Kornegay NM, Crews KR, Swanson H, Cheng C, Karol SE, Evans WE, Inaba H, Pui CH, Jeha S, Relling MV. Antibodies Predict Pegaspargase Allergic Reactions and Failure of Rechallenge. J Clin Oncol. 2019 Aug 10;37(23):2051-2061. doi:10.1200/JCO.18.02439. Epub 2019 Jun 12. PubMed PMID: 31188727.)Results: Is there any possibility that the level on day 6-8 of 2.8 was spurious? Were there any concomitant levels in this patient with this dose to make sense of such a high level?Response: this patient (UPN 11) after dose 3 of PEG had level of 2.8 on day 7. The scanned faxed report from AI Biotech was checked and confirmed the result of 2.8 as reported. We have no other levels after dose 3 to validate this result. After the preceding dose (#2) the result on day 7 was 0.78 and after the subsequent dose (#)4 the result on day 11 was 0.97. Based on the extreme deviance from a normal distribution we elected to remove this from the graphs and data analysis as discussed above. The one patient being described as having silent inactivation - I do not think I would characterize this as silent inactivation technically, since they had detectable activity levels at several time points, and no peak level is ever undetectable. I would consider this a transient accelerated clearance with dose 2 and 3. If silent inactivation was truly present, a patient should not have activity present for a full week and then have full activity with later doses. Patients with activity levels even as low as ~0.02 may still have asparagine depletion (AALL07p4).Response: It is our suggestion that accelerated clearance occurs through immune mechanism – and as we suggested above is similar to silent inactivation. Though it is possible that the patient had transient accelerated clearance with dose 2 and 3 as an explanation, it remains possible that that patient had transient silent inactivation after dose 3 and had become tolerant PEG when continued dose administered as we explain in the discussion paragraph 2 noting to Tong papers. It may be interesting to graph levels over time in some of these patients with very low levels. Was there ever more than one level obtained per dose? Were repeat levels obtained to verify the low activity levels or follow the activity level trend over time (as is suggested in some guidelines?). This would help characterize the PK better in these patients.Response: the patient UPN 28 with the silent activation did have level 0.05 day 8 then 0.00 day 16. It verified the very low levels. No others had confirmatory low levels. Was the choice to pre-medicate done at a strict, uniform point in time (i.e., was this a quasi-experimental design where a protocol to pre-medicate all patients was mandated?), or was pre-medication at the choice of the treating provider? If this was selected by the provider and not systematic, could there have been some slight selection bias where patients perceived to be at higher risk of hypersensitivity reactions were given pre-medication? This could have diminished the impact of pre-medication on reducing the rate of hypersensitivity reactions. Consider this in the discussion section if deemed relevant.As we suggested above these were mandated premedications given at a specified time. It was not at the choice of the treating provider; therefore bias seems unlikely.Discussion: I would more accurately call the silent inactivation here \"accelerated clearance\" as the patient had asparagine depletion for at least a week, even with the low level.We respectfully disagree as we consider accelerated clearance equal to silent inactivation, since they actually are the same mechanism. Consider discussing the possible value of obtaining activity levels in patients with questionable allergic reactions (grade 1/2). Response: Thank you. This was added to the last paragraph of the discussion. Could add as a limitation that it appears patients only had one activity level obtained per dose, which makes it challenging to interpret PK, especially in patients with higher or lower than expected activity levels. Response: Thank you, added to the limitation paragraph.Overall, a very well-done paper with logical conclusions. Thank you for allowing me to review the manuscript and for your hard work in this research. Is the work clearly and accurately presented and does it cite the current literature?Yes Is the study design appropriate and is the work technically sound?Partly Are sufficient details of methods and analysis provided to allow replication by others?Yes If applicable, is the statistical analysis and its interpretation appropriate?Partly Are all the source data underlying the results available to ensure full reproducibility?Yes Are the conclusions drawn adequately supported by the results?Yes References1. Cooper SL, Young DJ, Bowen CJ, Arwood NM, Poggi SG, Brown PA: Universal premedication and therapeutic drug monitoring for asparaginase-based therapy prevents infusion-associated acute adverse events and drug substitutions.Pediatr Blood Cancer. 2019; 66 (8): e27797 PubMed Abstract \| Publisher Full Text2. Marini BL, Brown J, Benitez L, Walling E, Hutchinson RJ, Mody R, Jasty Rao R, Slagle L, Bishop L, Pettit K, Bixby DL, Burke PW, Perissinotti AJ: A single-center multidisciplinary approach to managing the global Erwinia asparaginase shortage.Leuk Lymphoma. 2019. 1-15 PubMed Abstract \| Publisher Full Text3. Schore RJ, Devidas M, Bleyer A, Reaman GH, Winick N, Loh ML, Raetz EA, Carroll WL, Hunger SP, Angiolillo AL: Plasma asparaginase activity and asparagine depletion in acute lymphoblastic leukemia patients treated with pegaspargase on Children's Oncology Group AALL07P4.Leuk Lymphoma. 2019; 60 (7): 1740-1748 PubMed Abstract \| Publisher Full Text4. Park JH, Ritchie EK, Rao AV, Devlin S, Hsu M, Khawaja TT, O’Donnell B, Cazacu MI, Stopka DM, Tallman MS, Douer D: A Pediatric-Inspired Regimen Containing Multiple Doses of Intravenous Pegylated Asparaginase Appears Safe and Effective in Newly Diagnosed Adult Patients with Ph-Negative Acute Lymphoblastic Leukemia in Adults up to Age 60: Results of a Multi-Center Phase II Clinical Trial. Blood. 2016; 128 (1629)." } ] }, { "id": "53513", "date": "30 Sep 2019", "name": "Vinod Pullarkat", "expertise": [ "Reviewer Expertise Treatment of acute lymphoblastic leukemia" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript the authors have retrospectively examined the incidence of allergic reactions in a cohort of patients with ALL receiving multiple doses of PEGylated aspraginase. They show that premedication did not have an impact on allergic reactions. The study has drawbacks inherent in a retrospective analysis of a heterogeneously treated population with non standardized blood sampling as acknowledged by the authors. My comments are as follows:\nA concern with premedication is whether it will mask a true antibody mediated allergy. A major question therefore is whether the allergic reactions observed were actually antibody mediated allergic reactions or infusion reactions that do not affect asparaginase activity. Therefore authors should show the asparaginase levels of patients who had a reaction and compare to those who did not, in order to show whether the reactions observed are due to drug neutralizing antibodies.\n\nIn their conclusion (in the abstract) the authors focus on patients who receive only 2 doses of PEG asparaginase. In most regimens patients will receive more doses of PEG asparaginase and hence it is unclear why authors focus on patients who receive only 2 doses. The correct conclusion in my opinion is that premedication did not impact the incidence of allergic reaction and premedication did not mask silent inactivation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5115", "date": "17 Jan 2020", "name": "Bruce Bostrom", "role": "Author Response", "response": "Vinod Pullarkat: Department of Hematology and Hematopoietic Cell Transplantation, Gehr Family Center for Leukemia Research, City of Hope Medical Center, Duarte, CA, USA In this manuscript the authors have retrospectively examined the incidence of allergic reactions in a cohort of patients with ALL receiving multiple doses of PEGylated asparaginase. They show that premedication did not have an impact on allergic reactions. The study has drawbacks inherent in a retrospective analysis of a heterogeneously treated population with non-standardized blood sampling as acknowledged by the authors. My comments are as follows: A concern with premedication is whether it will mask a true antibody mediated allergy. A major question therefore is whether the allergic reactions observed were actually antibody mediated allergic reactions or infusion reactions that do not affect asparaginase activity. Therefore authors should show the asparaginase levels of patients who had a reaction and compare to those who did not, in order to show whether the reactions observed are due to drug neutralizing antibodies. Response: It would have been wonderful to be able to do this, but we cannot. Unfortunately, when patients had a reaction (be it allergic reaction or infusion reaction) no further pegaspargase was given. Therefore, these patients had no subsequent asparaginase activity measured. Additionally, this is an inherent difficulty with these patients – whose infusion is stopped right away and they never receive full dose of pegaspargase rendering subsequent asparaginase activity meaningless. In their conclusion (in the abstract) the authors focus on patients who receive only 2 doses of PEG asparaginase. In most regimens patients will receive more doses of PEG asparaginase and hence it is unclear why authors focus on patients who receive only 2 doses. The correct conclusion in my opinion is that premedication did not impact the incidence of allergic reaction and premedication did not mask silent inactivation. Response: We agree completely. The abstract and the paper has changes that completely address this. To the Abstract conclusion we added this sentence: “Though premedication resulted in lower allergic reactions the difference was not statistically different due to insufficient power.” In discussion the 1st paragraph completely addresses this: “Compared with historical controls that received similar therapy, premedication did reduce the incidence of grade 3 or 4 allergic reactions when measured per patient or per dose of pegaspargase. The power calculation was only 0.54 per patient and 0.24 per pegasparaginase dose, thus our study was underpowered to show statistical significance due to insufficient numbers.”The comment suggesting not monitoring levels in patients receiving only two doses of pegasparaginase is to potentially minimize unnecessary testing in this group of patients. In COG protocols only two doses of pegasparaginase are given to lower risk children who make up the majority of patients under the age of 18. However we have removed this recommendation due to large number of first dose pegaspargase levels missed in these patients. Is the work clearly and accurately presented and does it cite the current literature?Yes Is the study design appropriate and is the work technically sound?Yes Are sufficient details of methods and analysis provided to allow replication by others?Yes If applicable, is the statistical analysis and its interpretation appropriate?Partly Are all the source data underlying the results available to ensure full reproducibility?No Are the conclusions drawn adequately supported by the results?Partly Competing Interests: Speakers Bureau and Advisory board for Servier and Jazz pharmaceuticalsReviewer Expertise:Treatment of acute lymphoblastic leukemiaI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above." } ] }, { "id": "53445", "date": "01 Oct 2019", "name": "David J. Young", "expertise": [ "Reviewer Expertise Pediatrics", "pediatric hematology/oncology", "drug development", "clinical trials", "leukemia" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis report by Losasso, Bostrom and Messinger expand upon our growing understanding of the appropriate treatment with and management of asparaginase-based therapies in lymphoblastic leukemia. In this retrospective study, the authors have reviewed the single center experience of therapeutic drug monitoring, examining patient drug levels to determine the incidence of drug inactivation and looked at the relationship between pre-treatment and serious adverse events.\nIn their review, the authors found a 1.7% rate of drug inactivation. Of note, this rate is comparable to several different prior studies which the authors have referred here. Although the authors did not measure directly for inactivating antibodies, the use of drug activity levels to impute the presence of antibodies is now considered an acceptable approach as it is generally the primary determinant of drug activity for most patients, and as such is not a limitation in this work.\nTheir observation of transient inactivation and then recovery of drug efficacy is an important finding, and bears future examination as this strongly argues, as others have, for rechallenging patients with prior history, especially in high-risk patients where receiving multiple doses of asparaginase has been conclusively linked to better outcomes.\nContrary to other studies, the authors remark that they did not observe a significant decrease in clinically significant (grade 3-4) reactions with predication. Although statistically correct, it is difficult to support this statement for several reasons. First, although the authors do not see a statistically significant effect, there is without a doubt a clinically important trend of 50% reduction in events. Indeed, this would represent an absolute risk reduction of 8.7% with a number needed to treat of about 11, and is almost identical to other studies. Post hoc power analysis, although an admitted abuse of statistics, suggests that this data may have reached significance with 30-40 additional patients.\nFurthermore, the authors themselves continue to use and argue for premedication, which would argue that they agree that there is benefit to pre-medication despite the lack of statistical findings. Yet, the only reason for pre-medication, given the historic concerns regarding silent inactivation, is to reduce severe adverse reactions. These contradictions need to be resolved.\nFinally, the authors suggest that monitoring of drug levels is not necessary in low-risk patients, given that they did not identify inactivation during the first two doses. This statement is too strong. The authors themselves admit that there was a large number (83%) of missed levels during the first administration, likely a consequence of the admittedly chaotic logistics of early induction therapy experienced at many institutes. Although their data do suggest that silent inactivation following one dose would likely still be detectable at the second dose, this has not been rigorously tested, and is based primarily upon the experience of a single patient. Therefore, to include the recommendation of not testing low-risk patients, especially its inclusion within the abstract that (regrettably) may be all that some read, and without mentioning the rate of missed levels in the same abstract, is difficult to support.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5116", "date": "17 Jan 2020", "name": "Bruce Bostrom", "role": "Author Response", "response": "David Young Reviewer 3This report by Losasso, Bostrom and Messinger expand upon our growing understanding of the appropriate treatment with and management of asparaginase-based therapies in lymphoblastic leukemia. In this retrospective study, the authors have reviewed the single center experience of therapeutic drug monitoring, examining patient drug levels to determine the incidence of drug inactivation and looked at the relationship between pre-treatment and serious adverse events.In their review, the authors found a 1.7% rate of drug inactivation. Of note, this rate is comparable to several different prior studies which the authors have referred here. Although the authors did not measure directly for inactivating antibodies, the use of drug activity levels to impute the presence of antibodies is now considered an acceptable approach as it is generally the primary determinant of drug activity for most patients, and as such is not a limitation in this work.Their observation of transient inactivation and then recovery of drug efficacy is an important finding, and bears future examination as this strongly argues, as others have, for re-challenging patients with prior history, especially in high-risk patients where receiving multiple doses of asparaginase has been conclusively linked to better outcomes.Contrary to other studies, the authors remark that they did not observe a significant decrease in clinically significant (grade 3-4) reactions with predication. Although statistically correct, it is difficult to support this statement for several reasons. First, although the authors do not see a statistically significant effect, there is without a doubt a clinically important trend of 50% reduction in events. Indeed, this would represent an absolute risk reduction of 8.7% with a number needed to treat of about 11 and is almost identical to other studies. Post hoc power analysis, although an admitted abuse of statistics, suggests that this data may have reached significance with 30-40 additional patients.Furthermore, the authors themselves continue to use and argue for premedication, which would argue that they agree that there is benefit to pre-medication despite the lack of statistical findings. Yet, the only reason for pre-medication, given the historic concerns regarding silent inactivation, is to reduce severe adverse reactions. These contradictions need to be resolved. Response: we agree with the reviewer points and his astute observations. We would have included his Post hoc analysis if we could but were concerned like him about those who would view these statistics unkindly. Several things were altered: In Discussion 1st paragraph this sentence is now changed: “Compared with historical controls that received similar therapy, premedication did reduce the incidence of grade 3 or 4 allergic reactions when measured per patient or per dose of pegaspargase. The power calculation was only 0.54 per patient and 0.24 per pegasparaginase dose thus our study was underpowered to show statistical significance.” The 4th paragraph this whole section was removed: “Contrary to the findings of a large multi-institutional trial, where the introduction of premedication significantly reduced the incidence of high-grade allergic reactions, our study did not show a statistically significant reduction with the use of premedication. [7] Despite this, we continue to use premedication in all patients receiving pegasparaginase.”Finally, the authors suggest that monitoring of drug levels is not necessary in low-risk patients, given that they did not identify inactivation during the first two doses. This statement is too strong. The authors themselves admit that there was a large number (83%) of missed levels during the first administration, likely a consequence of the admittedly chaotic logistics of early induction therapy experienced at many institutes. Although their data do suggest that silent inactivation following one dose would likely still be detectable at the second dose, this has not been rigorously tested, and is based primarily upon the experience of a single patient. Therefore, to include the recommendation of not testing low-risk patients, especially its inclusion within the abstract that (regrettably) may be all that some read, and without mentioning the rate of missed levels in the same abstract, is difficult to support. Response: we do agree with the reviewer completely that the number of missed levels after the 1st dose is very high. We also agree that our numbers are too small for this strong conclusion. We have altered the abstract to include this sentence in the results: “Asparaginase dose levels were mostly missed after the first asparaginase dose.” We eliminated this sentence: “No patient had silent inactivation after the first pegaspargase dose.” And the conclusions: “We found low incidence of silent inactivation with intravenous pegaspargase. Future studies may confirm if patients treated with regimens with only two doses of pegasparaginase, given with dexamethasone, may not need asparaginase levels due to even lower silent inactivation.” In discussion 2nd paragraph this was altered: “No decrease in pegaspargase activity was seen in standard risk patients who received only two doses of pegaspargase in combination with oral dexamethasone. This observation is limited by small numbers of patients and multiple missed levels after the first pegaspargase dose.” The 4th paragraph was altered also: “We found low incidence of silent inactivation with intravenous pegaspargase. Our study suggests that patients treated with regimens that include only two doses of pegasparaginase, given with dexamethasone, may not need asparaginase levels due to even lower silent inactivation, but this would need confirmation by larger studies.” Is the work clearly and accurately presented and does it cite the current literature?Yes Is the study design appropriate and is the work technically sound?Yes Are sufficient details of methods and analysis provided to allow replication by others?Yes If applicable, is the statistical analysis and its interpretation appropriate?Yes Are all the source data underlying the results available to ensure full reproducibility?Yes Are the conclusions drawn adequately supported by the results?Partly Competing InterestsNo competing interests were disclosed." } ] } ]	1	https://f1000research.com/articles/8-1007
https://f1000research.com/articles/8-1904/v1	11 Nov 19	{ "type": "Research Article", "title": "Making the invisible visible: the availability and desirability of adherence data in routine CF care– findings from a national questionnaire survey", "authors": [ "Louisa Robinson", "Chin Maguire", "Zhe Hui Hoo", "Martin J. Wildman", "Louisa Robinson", "Chin Maguire", "Zhe Hui Hoo" ], "abstract": "Background: Inhaled medications for cystic fibrosis (CF) are effective but adherence is low. Clinicians find it difficult to estimate how much treatment people with CF (PWCF) take, whilst objective adherence measurement demonstrates that patients are poorly calibrated with a tendency to over-estimate actual adherence. The diagnostic approach to a PWCF with deteriorating clinical status and very low adherence is likely to be different to the approach to a deteriorating patient with optimal adherence. Access to objective adherence data in routine consultations could help to overcome diagnostic challenges for clinicians and people with CF. Attitudes of clinicians to the use and importance of routinely available adherence data is unknown. Methods: We conducted an online questionnaire survey with UK CF centres. We asked five questions relating to the current use and perception of objective measurements of adherence in routine care. Results: A total of eight CF centres completed the questionnaire. Few of the responding centres have adherence data readily available in routine clinics (13% of centres use medicines possession ratio; of centres with access to I-nebs® it was estimated that 17% of patients had I-neb data regularly available in clinics). All centres considered the availability of objectively measured adherence data to be important. Respondents identified that systems developed to provide adherence data in clinical practice must provide data to both clinicians and patients that is readily understood and easy to use. Conclusions: Centres perceived the availability of adherence data in routine care to be important but objective measures of adherence is rarely available at present.", "keywords": [ "cystic fibrosis", "medication adherence", "routine monitoring", "nebulisers" ], "content": "Introduction\n\nCystic Fibrosis (CF) is a multi-system life-limiting genetic condition in which the most common cause of death is respiratory failure. Daily use of inhaled mucolytic and antibiotic medications are effective to prevent pulmonary exacerbations and preserve lung function1,2. Low adherence to CF medications is associated with poorer health outcomes3, yet real-world adherence to inhaled medications is only 35–50%4,5.\n\nAnother problem is the potential for diagnostic uncertainties – in a study where the objectively measured adherence was 36%, people with CF reported median adherence of 80% whilst clinicians estimated adherence rate of 55–60%5. This disparity makes it challenging for healthcare professionals to make informed clinical decisions. The ‘invisibility’ of adherence in routine care can result in consultations characterised by the ‘lamp post syndrome’6, whereby there is a tendency for clinicians to use readily available information which can be misleading. In other words, clinicians ended up seeking information “where the light is” rather than to use all relevant data sources. Since neither patient self-report nor clinician estimation provides an accurate indication of medication adherence, adherence will be invisible in those centres without systems that make objectively measured adherence visible. This lack of adherence data is critical for diagnosis and assessment. Indeed, the approach in a patient with deteriorating clinical status and low adherence (where treatment failure is due to non-use of existing treatments) is very different to the approach in a patient with deteriorating clinical status and high adherence (where existing treatments have failed, and treatment escalation must be considered).\n\nFurthermore, enabling self-monitoring using objective feedback is associated with increases in target health behaviours7 and has been identified as a facilitator in nebuliser adherence8. Access to, and feedback from, accurate adherence data could facilitate more effective self-monitoring and self-care for people with CF.\n\nDespite evidence pointing to the importance of objectively measured adherence data in routine CF care, it remains unclear how frequently clinicians readily have access to objective adherence data in their day-to-day practice and whether such access is desired by clinicians. We therefore conducted an online survey among UK CF centres to establish the perceived importance of routinely available adherence data, and the extent to which this data is currently used in routine care.\n\n\nMethods\n\nCentre directors from all 29 UK adult CF centres were invited by email on 12 March 2019 to participate in a short online questionnaire about their views and practices in using adherence data in their day-to-day management of people with CF. Where available, contact details were sought through the CF registry; in the absence of this, email addresses were ascertained through hospital websites.\n\nCentre directors were encouraged to discuss the questions with their clinical colleagues before responding. Centres that did not respond after two weeks were sent two reminders, at two week intervals. All responses were collected within two weeks of the final reminder.\n\nThe participant information sheet detailed that responses to the survey would be used for research purposes. The information sheet was embedded within the invitation email. Consent to participate was implied through completion of the questionnaire. The questionnaire was designed with Qualtrics© 2019 software (version March 2019, Provo, Utah) and consisted of five-items with a mixture of Likert scale, percentage estimate and free-text responses (see Extended data). Respondent Internet Protocol (IP) addresses were captured and used to check for duplicate responses. No duplicate responses were captured.\n\nResponses to Likert scale questions (Q1, Q4) were coded 1–5 (1= Very important, 5= Not important) and medians and inter-quartile ranges summarised. For responses to questions requesting a percentage estimate (Q2, Q3), means and standard deviations were calculated. Free-text responses (Q5) were summarised by extracting key themes.\n\nThe study received approval from the School of Health and Related Research (ScHARR) Research Ethics Committee, University of Sheffield (ref: 024042). The survey was hosted on Qualtrics (survey tool approved by the University of Sheffield Corporate Information and Computing Services). The University of Sheffield was the data controller and all survey data exported for analysis was stored on an access restricted folder on the University shared file store.\n\n\nResults\n\nA total of eight adult UK CF centres (28%) provided data from sites across England, Northern Ireland and Wales. Summaries by questionnaire item are summarised in Table 1.\n\nAn average of 87% of patients using inhaled therapies were estimated not to have up to date medicines possession ratio or pharmacy refill data available during a typical outpatient clinic. This included four centres that said 100% of their patients would not have this information available. Across five centres, an average of 83% of patients were estimated not to have I-neb® data readily available at the point of consultation. It was stated by two sites that I-nebs were not used at their centre and it is important to recognise that at the time the study, the only routinely available devices to collect objective adherence data were Inebs®.\n\nIn contrast to low availability of objective adherence data, all centres considered having up-to-date, objective adherence data for inhaled therapies at the point of diagnosis to be at least moderately important; 57% considered this to be important and a further 29% considered this to be very important. When asked if they thought that a system able to automatically collect and provide objective, up-to-date adherence data within a consultation would be important, all responding centres said that this would be important to have in their centre and 50% of respondents felt this would be very important.\n\nA total of seven free-text responses were received for question five regarding the key features for a system to routinely collect objective adherence data. Multiple respondents stated that “ease of use” would be important, as well as quick and reliable data access. Centres also stated that it would be important for data to be available for both patients and staff to facilitate discussions on adherence.\n\n\nConclusion\n\nAdherence is important for accurate diagnosis, treatment planning and self-management in people with CF3,6,8. We have demonstrated that whilst centres providing care to people with CF perceive objective adherence data to be important, comprehensive provision of these data is not a feature of current CF clinical practice. A small percentage of people with CF have objective measures of adherence available for consultations (between 13–17% depending on the use of medication possession ratio or I-neb®). This is despite an overall consensus that access to objective adherence data is important for making accurate diagnoses. If a system could produce up-to-date, routinely collected objective adherence data for use in consultations, all centres felt this would be an important asset.\n\nThe results of this study demonstrate that in the responding centres clinicians felt that objectively measured adherence at the point of consultation would be useful but was rarely available. A limitation of the current study is that with only 8 out of 29 UK adult centres responding, it is uncertain whether objective adherence data is considered important across the whole of the UK. Nevertheless, it is notable that within the centres that did respond, there was a consensus that objective adherence data was important and no centres that considered objective adherence data to be unimportant.\n\n\nEthical approval\n\nEthical approval for the collection and analysis of data was obtained from School of Health and Related Research (ScHARR) Research Ethics Committee, University of Sheffield (reference: 024042)\n\n\nData availability\n\nORDA: Findings from a national questionnaire survey of UK CF centres on availability and desirability of routinely available adherence data, https://doi.org/10.15131/shef.data.10059251.v19.\n\nThis project contains the following underlying data:\n\n- CSV file exhibiting the responses to the questionnaire for the eight centres\n\nORDA: Findings from a national questionnaire survey of UK CF centres on availability and desirability of routinely available adherence data, https://doi.org/10.15131/shef.data.10059251.v19.\n\nThis project contains the following extended data:\n\n- Questionnaire sent to the CF centers\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nRyan G, Mukhopadhyay S, Singh M: Nebulised anti-pseudomonal antibiotics for cystic fibrosis. In: Ryan G, editor. Cochrane Database Syst Rev. Chichester, UK: John Wiley & Sons, Ltd. 2003; (3): CD001021. PubMed Abstract \| Publisher Full Text\n\nRyan G, Singh M, Dwan K: Inhaled antibiotics for long-term therapy in cystic fibrosis. Ryan G, editor. Cochrane Database Syst Rev. Chichester, UK: John Wiley & Sons, Ltd. 2011; (3): CD001021. PubMed Abstract \| Publisher Full Text\n\nEakin MN, Bilderback A, Boyle MP, et al.: Longitudinal association between medication adherence and lung health in people with cystic fibrosis. J Cyst Fibros. 2011; 10(4): 258–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQuittner AL, Zhang J, Marynchenko M, et al.: Pulmonary medication adherence and health-care use in cystic fibrosis. Chest. 2014; 146(1): 142–51. PubMed Abstract \| Publisher Full Text\n\nDaniels T, Goodacre L, Sutton C, et al.: Accurate assessment of adherence: self-report and clinician report vs electronic monitoring of nebulizers. Chest. 2011; 140(2): 425–32. PubMed Abstract \| Publisher Full Text\n\nWildman MJ, Hoo ZH: Moving cystic fibrosis care from rescue to prevention by embedding adherence measurement in routine care. Paediatr Respir Rev. W.B. Saunders. 2014; 15 Suppl 1: 16–8. PubMed Abstract \| Publisher Full Text\n\nBurke LE, Wang J, Sevick MA: Self-monitoring in weight loss: a systematic review of the literature. J Am Diet Assoc. Elsevier. 2011; 111(1): 92–102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArden MA, Drabble S, O’Cathain A, et al.: Adherence to medication in adults with Cystic Fibrosis: An investigation using objective adherence data and the Theoretical Domains Framework. Br J Health Psychol. John Wiley & Sons, Ltd. 2019; 24(2): 357–380. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobinson L, Maguire C, Wildman M, et al.: Findings from a national questionnaire survey of UK CF centres on availability and desirability of routinely available adherence data. figshare. Dataset. 2019. http://www.doi.org/10.15131/shef.data.10059251.v1" }	[ { "id": "57625", "date": "30 Dec 2019", "name": "Dominique Pougheon Bertrand", "expertise": [ "Reviewer Expertise Public Health", "Quality Improvement", "Patient Experience", "Qualitative methodology (grounded theory)", "Cystic Fibrosis." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article deals with the adherence of adult patients with CF to the nebulization treatments and more particularly the importance of assessing their adherence in clinic consultations to establish a good diagnosis of the patient’s health (a deterioration might be due to non-adherence to a prescribed treatment or other causes despite good adherence to the treatments) and thus to offer the best treatment strategies to the patient.\n\nThe purpose of the study is to assess the availability and desirability, for the clinicians involved in CF care in the UK, of objective adherence data from their patients. Five questions were asked through an online questionnaire survey to the UK Adult CF centres. Availability refers to Medical Possession Ratio (MPR - refill data) or I-neb data (a device used to nebulize various treatments) available during the clinic visit. Desirability refers to the value that clinicians place on this data at the point of consultation.\n\nThough clinicians place importance on objective adherence data during the clinic visits of the patients, this data is rarely available (only available for about 15% patients) and their provision not a feature of CF care in current clinical practice. The author indicates as a limitation of the study the fact that the response rate to the survey is only 28% of the CF centers (8 centers out of 29).\n\nThis paper is very clear, the methods is presented in details so it could be replicated in another study, and the results are fully presented. It gives perspectives to improve both the quality of care and the health outcomes of the patients by increasing the availability and use of patient adherence data to nebulized treatments. This would increase the relevance of prescriptions and possibly open up a discussion with the patient about the feasibility of their treatments and routines necessary to sustain care in daily life.\n\nMay the author consider citing the following paper (6th position in the reference) as it is an assessment of adherence in CF patients for inhaled therapies in France: Heloïse Rouzé et al.1\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "58100", "date": "03 Jan 2020", "name": "Mark Hew", "expertise": [ "Reviewer Expertise Evidence based health care", "severe asthma", "allergic disease", "pleural disease" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes a survey of adherence monitoring availability, and views regarding the benefits of such, in the context of UK national CF centres. Aims, methods, results are described in sufficient detail for the reader to understand and follow. The discussion is reasonable and does not exceed the scope of the data. The results demonstrate substantial limitations in clinicians’ ability to capture and utilise adherence data in clinical care- findings important to highlight and necessary to address.\nI have the following major comments:\nThe 8/29 response rate from centre directors is disappointing. This may therefore represent significant selection bias and this bias should be explicitly mentioned in the limitations; i.e. centres with less interest in an adherence monitoring system are less likely to respond.\n\nThe sub-optimal response rate may also indicate limited interest or engagement among some centres for implementing a concerted adherence monitoring system. The authors might consider including this in their discussion, in a sensitive and tactful manner.\n\nWhile the respondents agreed a user-friendly system to make adherence data available in consultations was desirable, they were not asked what barriers currently stood in the way of implementing this- (staff, equipment, time). If this survey is repeated in future, this would yield useful information to develop and implement such a system.\n\nAs a practitioner outside the CF field, I am surprised that adherence monitoring is not a routine part of routine assessment, especially prior to the use of eye-wateringly expensive advanced therapies. For example, the Severe Asthma centres in the UK are tasked with ensuring objective adherence data is acceptable prior to commencing biologics (far less expensive than gene potentiators), and the Australian Pharmaceutical benefits system requests that adherence is addressed prior to prescription of biologics (although objective measurement is not a requirement here). While clinicians are naturally (and, I judge, correctly) reluctant to set adherence thresholds requirements prior to implementing advanced therapies, knowledge of adherence patterns is always desirable when contemplating stepping-up any therapy. The authors may consider drawing comparisons between the practice in CF and severe asthma fields even in the UK alone.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5165", "date": "17 Jan 2020", "name": "Louisa Robinson", "role": "Author Response", "response": "Dear Professor Hew,Please see our responses to your comments below and refer to the revised article which has now been published.1. The 8/29 response rate from centre directors is disappointing. This may therefore represent significant selection bias and this bias should be explicitly mentioned in the limitations; i.e. centres with less interest in an adherence monitoring system are less likely to respond.We agree this is an important bias and have now included this in the new 3rd paragraph of the conclusion. The sentence now reads: \"Centres with less interest in monitoring adherence may be less likely to respond, so it is uncertain whether objective adherence data is considered important across the whole of the UK.\"2. The sub-optimal response rate may also indicate limited interest or engagement among some centres for implementing a concerted adherence monitoring system. The authors might consider including this in their discussion, in a sensitive and tactful manner.We agree this is an important point and have now added this in the 1st paragraph of the conclusion. The sentence now reads: \"Only a small percentage of people with CF have objective measures of adherence available for consultations (between 13–17% depending on the use of medication possession ratio or I-neb®) with this figure likely to be even lower among centres which did not respond to the survey.\" 3. While the respondents agreed a user-friendly system to make adherence data available in consultations was desirable, they were not asked what barriers currently stood in the way of implementing this- (staff, equipment, time). If this survey is repeated in future, this would yield useful information to develop and implement such a system.This is a good suggestion and we have now added the following sentence in the 2nd paragraph of the conclusion: \"To help embed an objective adherence monitoring system within routine CF clinical practice, it would be useful to ascertain the barriers for implementing such a system among CF centres in future surveys.\" 4. As a practitioner outside the CF field, I am surprised that adherence monitoring is not a routine part of routine assessment, especially prior to the use of eye-wateringly expensive advanced therapies. For example, the Severe Asthma centres in the UK are tasked with ensuring objective adherence data is acceptable prior to commencing biologics (far less expensive than gene potentiators), and the Australian Pharmaceutical benefits system requests that adherence is addressed prior to prescription of biologics (although objective measurement is not a requirement here). While clinicians are naturally (and, I judge, correctly) reluctant to set adherence thresholds requirements prior to implementing advanced therapies, knowledge of adherence patterns is always desirable when contemplating stepping-up any therapy. The authors may consider drawing comparisons between the practice in CF and severe asthma fields even in the UK alone.This is a very important point and we have now drawn such a comparison in the 2nd paragraph of the conclusion: \"The situation in CF contrasts with that of other long-term respiratory conditions such as asthma whereby adherence measurement is mandated prior to commencing expensive biologics e.g. mepolizumab.\"" } ] } ]	1	https://f1000research.com/articles/8-1904
https://f1000research.com/articles/9-23/v1	16 Jan 20	{ "type": "Research Article", "title": "Cytomorphological patterns of thyroid lesions among 1646 Sudanese patients: what we can learn from fine needle aspiration cytology retrospective analysis?", "authors": [ "Ali I. E. Osman", "Ahmed O. Almobarak", "Asma Kamalaldin Mohammed", "Nouh S. Mohamed", "Mohamed S. Muneer", "Ammar B. Omer", "Hussam M. A. Ibrahim", "Emmanuel E. Siddig", "Eman T. Ali", "Abdalla Munir", "Ali M. M. Edris", "Eiman S. Ahmed", "Lubna S. Elnour", "Rowa Hassan", "Ali I. E. Osman", "Ahmed O. Almobarak", "Asma Kamalaldin Mohammed", "Nouh S. Mohamed", "Mohamed S. Muneer", "Ammar B. Omer", "Hussam M. A. Ibrahim", "Eman T. Ali", "Abdalla Munir", "Ali M. M. Edris", "Eiman S. Ahmed", "Lubna S. Elnour", "Rowa Hassan" ], "abstract": "Introduction: Fine-needle aspiration (FNA) cytology biopsy of the thyroid gland is an accurate and useful diagnostic tool in the initial evaluation of nodular thyroid lesions. We aimed in this study to determine the cytomorphological patterns of thyroid lesions diagnosed by FNA among Sudanese patients. Methods: A descriptive retrospective, clinic-based study was performed. Cytopathological records of patients that attended the Total Lab Care Clinic in Khartoum-Sudan between January 2016 and December 2017 were reviewed. Results: A total of 1646 patients records were reviewed; 1385 (84.1%) were females and 261 (15.9%) males. A total of 1563 (94.9%) were negative for malignancy, 39 (2.4%) were positive for malignancy, 42 (2.6%) were indeterminate for malignancy and 2 (0.1%) were non-diagnostic. Colloid goiter was seen in 1147 patients (73.4%), benign hemorrhagic cysts were seen in 257, Hashimoto thyroiditis was seen in 77, benign thyroid nodules were seen in 76, keratocysts were seen in 2, thyroglossal duct cysts were seen in 2, thyroid follicular adenoma was seen in 1 and myxedema was seen in 1. For malignant patients; 11 had anaplastic thyroid cancer, 8 had papillary thyroid cancer, 7 had follicular thyroid cancer, 5 had metastatic thyroid cancer, 4 had medullary thyroid cancer, 3 had non-Hodgkin lymphoma and 1 had thyroid follicular adenoma. For those indeterminate for malignancy, 24 had follicular neoplasm and 18 had Hurthle cell neoplasm. Conclusions: Fine needle aspiration cytology for thyroid nodules provides a rapid and non-invasive technique for the evaluation and differentiation between benign and malignant lesions. This study also addresses the increased predominance of benign thyroid lesions among young patients and thyroid malignancy among the 4th decade of life.", "keywords": [ "Thyroid nodules", "Cytomorphological features", "Fine Needle Aspiration", "Sudan." ], "content": "Introduction\n\nFine-needle aspiration cytology (FNA) biopsy of the thyroid gland is an accurate and useful diagnostic tool in the initial evaluation of the nodular thyroid lesions1–4. Since ≤5% of thyroid nodules are malignant, most thyroid swellings are non-neoplastic and may not require surgical intercession5–7. Although surgical management of thyroid nodules is a feasible option, it is associated with many adverse effects, including development of hypoparathyroidism and an enduring need for thyroid hormone replacement therapy8, and also risk during surgical intercession such as injury of the recurrent nerves, bacterial infections of the surgery wound, and other related factors9. Therefore, to avoid unnecessary thyroid surgery, the standardized Bethesda scoring system and the ACR Thyroid Imaging Reporting and Data System (ACR TI-RADS) have been developed to empower clinicians to perform suitable therapeutic intercessions10,11. When an accurate preoperative diagnosis can be made, and no subsequent risk of malignant changes was suspected, surgery can be avoided12,13. FNA is one of the initial preoperative screening procedures for the diagnosis of nodular thyroid disease and considered as the most accurate diagnostic modality4,13–16, with a sensitivity ranging between 43 to 95%, and specificity of 47 to 100% for thyroid lesions16,17. This wide range of sensitivity and specificity is attributed to the testing of suspicious and rare cases and the inclusion of occult papillary carcinoma in the category of false negative diagnosis18,19. Also, FNA is a safe and useful procedure for the differential diagnosis of thyroid malignancies, such as medullary thyroid carcinoma20.\n\nIn Sudan, ultrasound-guided FNA is considered to be an expensive technique in order to be either provided by the hospital or requested by the physician managing the patient. Lacking the epidemiological data addressing the prevalence of thyroid malignancy in Sudan, besides, there is no recent descriptive study updating the status of thyroid malignancy through the past six years. Therefore, this study aimed at determining the cytomorphological patterns of thyroid lesions diagnosed by FNA among Sudanese patients attending a cytology clinic in Khartoum state, Sudan from January 2016 to December 2017.\n\n\nMethods\n\nA descriptive retrospective, clinic-based study in which we reviewed records of all patients referred to the Thyroid Nodule Center at the Total Lab Care Clinic in Khartoum state, Sudan for the evaluation of nodular thyroid disease from January 2016 to December 2017. Other than the presence of thyroid disease, no other eligibility criteria were used. The retrospective data, including type of disease, macroscopic appearance and malignancy status, were collected from the cytopathological records and sorted synonymously for analysis.\n\nFNA was performed previously by two expert physicians using the palpation technique21. In cases of multinodular lesions and complex nodules consisting of solid and fluid matters, FNA was done multiple times to avoid sampling from single nodule or aspirating cystic fluid only. Uninodular lesions with solid composition were aspirated four times to avoid bias due to sampling from single area and to insure coverage of the whole nodule. Meanwhile, in case of small nodules which were difficult to palpate (<1 cm), cystic nodules and/or nodules located posteriorly in the thyroid gland, ultrasound guided FNA were done by experienced radiologist using a LOGIQ P5 Trackball device (Guangzhou Rongtao Medical Technology Co., Ltd, China). Four smears for each patient were taken from different regions of the thyroid nodule in order to prevent misdiagnosis. The four FNA smears of each patient were fixed in 95% ethanol according to procedures outlines by Safneck et al.22. Then, two smears were stained using Papanicolaou (Pap) stain and the other two were stained using May–Grünwald Giemsa (MGG) stain, as described previously23,24. Cytopathologic diagnosis was performed by two experienced cytopathologists using microscopy with a high-power magnification lenses (X40). Both cytopathologists’ results were compared with each other to confirm the diagnosis. Results of diagnosis of smears were categorized as non-diagnostic if insufficient cellular material was present and no evidence of cellular atypia was found (<6 groups of cells containing ≥15 cells each), and into negative for malignancy, positive for malignancy and indeterminate for malignancy according to the Bethesda System for Reporting Thyroid Cytopathology10.\n\nA total of 1646 patients were identified and their smears were reviewed. The main cytomorphological features used for classification of thyroid nodules used included; cellularity of smears, cellular architectures (microfollicles and macrofollicles), cohesiveness, nuclear to cytoplasmic ratio, intactness of cell membrane, size of nuclei, pleomorphism, shape of nuclei, condensation of chromatin, alteration of nuclear contours, prominence of conspicuous nucleoli, and presence of pseudo-inclusions, nuclear grooves, and nuclear enlargement. Additionally, the presence or absence of Hurthle cells, amyloid, colloid, psammoma bodies, inflammatory cells, necrotic material, and tumor diathesis. The criteria of the Bethesda System for Reporting Thyroid Cytopathology were followed25.\n\nDescriptive data were analyzed using the Statistical Package for the Social Sciences (SPSSv16). Pearson Chi Square Test was used to test the relationship between the different categorical variables. P value < 0.05 was considered a statistically significant.\n\nEthical approval was obtained from the ethics committee of the Faculty of Medical Laboratory Science, University of Khartoum-Sudan (UofK, FMLS 01/2017). Written informed consent for the procedure and for use of patient data for publication were obtained previously by the Thyroid Nodule Center at the Total Lab Care Clinic.\n\n\nResults\n\nThe study population consisted of 1385 (84.1%) females with mean age of 42.46±14.19, and 261 (15.9%) males with mean age of 48.29±16.84. The male to female ratio was 1:5.3. Age of patients was grouped based on interval of 10 years, age group with peak thyroid lesions was 31 to 40 years followed by 41 to 50 years.\n\nBased on the Bethesda system, results of both cytopathologists were compared with each other to obtain the final diagnosis. Each smear was diagnosed by the first cytopathologist was matching to the diagnosis of the second cytopathologist. The retrospective results of the cytomorphological features of the FNA and data recorded about the size of swellings, site of swelling distributed based on age group were shown in Table 1. Size of swilling was significantly associated with malignancy (P=0.006), whereas site of swelling was not. Individual results for each patient are available as Underlying data26.\n\n\nPatient history of thyroidectomy and family history of thyroid disease\n\nIn total, 13/1646 patients only reported past medical history of thyroidectomy of one thyroid lobe, while the remaining 1633/1646 were not having any previous surgical removal of any of the thyroid lobes.\n\nFamily history of thyroid diseases were only reported among the negative and positive for malignancy, 77/1646 and 39/1646 respectively. Having family history of thyroid diseases was statistically significant (p = 0.0001).\n\nFamily history with thyroid diseases among patients diagnosed with malignancies was mostly noted among anaplastic carcinoma 11/39, followed by papillary thyroid cancer and follicular thyroid cancer (8/39 and 7/39, respectively). However, the remaining malignancies were also reported: 5/39 patients with metastatic cancer, 4/39 with medullary thyroid cancer and 3/39 patients with non-Hodgkin lymphoma.\n\nPatients positive for malignancy were classified as follows: 11 (28.2%) had anaplastic thyroid cancer, 8 (20.5%) papillary thyroid cancer, 7 (17.9%) follicular thyroid cancer, 5 (12.8%) metastatic thyroid cancer, 4 (10.2%) medullary thyroid cancer, 3 (7.8%) non-Hodgkin lymphoma and 1 (2.6%) patient with thyroid follicular adenoma presenting malignant cells. Patients aged 61–70 years showed higher frequency of malignancy (Table 2).\n\nIn patients indeterminate for malignancy, the most frequent ages were 21 to 50 years, the peak age group was between the 20–39 years. The FNA diagnosis of the indeterminate for thyroid malignancy were grouped into two diagnostic verdicts, 24 (57.1%) as follicular neoplasm and 18 (43.9%) as Hurthle cell neoplasm. Figure 1 shows the different cytomorphological patterns of the thyroid lesions diagnosed in this study.\n\n(A) Monolayer sheets of follicular cells from patient with goiter (Giemsa stain, X100). (B) Follicular neoplasm with Hurthle cells. (C) Papillary thyroid carcinoma with flat sheet that showed nuclear grooves (Giemsa stain, X40). (D) smear showed medullary thyroid carcinoma note the single cells with plasmacytoid and spindle cells differentiation with scant cytoplasm and coarsely granular chromatin (Papanicolaou, X40). (E) Anaplastic carcinoma showed bizarre and giant malignant cells with marked pleomorphic cells (Giemsa stain, X40). (F) Non-Hodgkin’s lymphoma (Giemsa stain, X40).\n\n\nDiscussion\n\nThyroid nodules are a common clinical problem27. FNA of the thyroid is practiced worldwide and is reliable and cost-effective diagnostic procedure to diagnose thyroid lesions that may need surgical excision or conservative management20,28. The key factors to ensure informative thyroid FNA are having an adequate or representative cell sample and expertise of the healthcare professional performing the thyroid cytology4,29,30.\n\nThyroid cancer is the most common endocrine cancer (approximately 1.0%–1.5% of all new cancers diagnosed each year)31 and its incidence has continuously increased in the last three decades all over the world, including Sudan32–34. In this study, thyroid cancers were seen predominantly among those aged 30–59 years with the peak age in those aged 50–59 years. These results are in agreement with previous reports35,36. In this study, most of the diagnosed thyroid lesions were found to be benign in nature. However, in another study conducted by Caruso and his colleagues, they reported a lower percentage (74%) of benign thyroid lesions in more than 9000 patients37. Gharib et al.38, after reviewing 11,000 specimens, found 69% of examined thyroid lesions were benign lesions. Although benign nodules were reported to display morphologic changes over time, these were referred to as degenerative or mummified nodules30,39,40. This observation can lead to a non-diagnostic result on FNA, which could increase the incidence of unnecessary thyroid surgery39,41. Remarkably, the higher rate of colloid goiter among the benign lesions detected in this study can be related to the following reasons as discussed in the literature42,43: iodine deficiency, overgrowth of normal thyroid tissue, thyroid cyst, chronic inflammation of the thyroid (thyroiditis), multinodular goiter, thyroid cancer. In the current study, the relatively higher frequency of the colloid goiter from all retrieved thyroid lesions was in parity with previous reports conducted in Sudan30,44,45. Also, these results were in concordance with Nadira et al.46; which shows that colloid goiter represent about 77.2% of the thyroid lesions. Additionally, Hadi et al.47 revealed quite similar results of colloid goiter in 66.6% of the total cases.\n\nRegarding thyroiditis, Hashimoto thyroiditis had the highest prevalence. Hashimoto thyroiditis is a common autoimmune disease characterized by marked lymphoid infiltrate destroying the thyroid follicles; it has a peak incidence between 40 and 60 years of age and a female predominance10,48. In the present study, Hashimoto thyroiditis were common among patients falling in the age between 21 and 50 years. But, Bhatia and his colleagues observed the commonest age group is those 20–39 years of age49. Also, female predominance has been observed in this study which agrees with previous published studies48,50–52.\n\nWith respect to malignant thyroid lesions, nearly 3% of the study cases were diagnosed as malignant and the majority of these were in females. Moreover, the malignant cases were reported to be in those between 30 and 59 years of age. Although anaplastic carcinoma occurs in approximately 2% of reported thyroid cancer cases53, this study depicted that anaplastic carcinoma accounted the highest proportion of thyroid malignancies followed by papillary thyroid carcinoma and follicular thyroid carcinoma in Sudan. This result is comparably different from other studies showed higher prevalence of thyroid malignancy34,54.\n\nOur study has highlighted several potential benefits of FNA which is consistent with the previous studies addressing the FNA reimbursements4,38,55,56. The most important benefits are that FNA is a simple, safe and cost-effective first-line method to investigate thyroid lesions, particularly in low-resource settings such as Sudan, in which most of the patients suffering from thyroid lesions presented late to the clinics due to their low-income status. Furthermore, the diagnosis of benign lesions is 50-fold that of malignant ones; this can be interpreted as an increase in the community awareness about thyroid diseases as well as more clinicians recognizing the utility of FNA in evaluating thyroid nodule57,58. However, further studies considering this increase in benign lesions showed be well investigated in respect to all factors associated with developing benign thyroid lesions.\n\n\nLimitations\n\nMost of the patients underwent surgical removal in different clinics. Therefore, follow-up data of the negative for malignancy lesions that showed non-neoplastic features were not correlated with the clinical presentation and ultrasonographic findings to conclude the final diagnosis. Follow up data of the repeated palpation, FNA and ultrasound at 6–18 months intervals to detect the appearance of significant growth or suspicious sonographic changes were not feasible to obtain from all the study participants and were excluded to avoid bias.\n\n\nConclusions\n\nThis study showed the usefulness of FNA in the evaluation of thyroid lesions and a high diagnostic performance for detection and differentiation of benign lesions from malignancy, with low rates of technical failures and complications with respect to patients’ economic status since ultrasound guided FNA were only used for unpalpable nodules. Also, this study addresses the increased predominance of benign thyroid lesions among young patients and thyroid malignancy among those aged 30–39.\n\n\nData availability\n\nHarvard Dataverse: Cytomorphological Patterns of Thyroid Lesions among 1646 Sudanese Patients: What we can learn from Fine Needle Aspiration Cytology Retrospective Analysis? https://doi.org/10.7910/DVN/AYEZVP (version 2)26.\n\nThis project contains the following underlying data:\n\nThyroid data (underlying data, including demographic details and information about malignancy, in SAV format).\n\nThyroid research (2) (underlying data in XLSX format).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe are of great thanks for kind collaboration and assistance of the clinical staff of Thyroid Nodule Clinic at the Total Lab Care Clinic, Khartoum, Sudan during slide examination and interpretations. Great thanks to all participants contributed to this work.\n\n\nReferences\n\nMazzaferri EL: Management of a solitary thyroid nodule. N Engl J Med. 1993; 328(8): 553–559. PubMed Abstract \| Publisher Full Text\n\nHegedüs L: Clinical practice. The thyroid nodule. N Engl J Med. 2004; 351(17): 1764–1771. PubMed Abstract \| Publisher Full Text\n\nSiddig E: Fine needle aspiration: past, current practice and recent developments. Biotech Histochem. 2014; 89(4): 241–244. PubMed Abstract \| Publisher Full Text\n\nCan AS, Peker K: Comparison of palpation-versus ultrasound-guided fine-needle aspiration biopsies in the evaluation of thyroid nodules. BMC Res Notes. 2008; 1(1): 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSukumaran R, Kattoor J, Pillai KR, et al.: Fine needle aspiration cytology of thyroid lesions and its correlation with histopathology in a series of 248 patients. Indian J Surg Oncol. 2014; 5(3): 237–241. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nŁącka K: Rare thyroid diseases. In: Thyroid research: 2013. BioMed Central. 2013; 6: A36. Reference Source\n\nSclabas GM, Staerkel GA, Shapiro SE, et al.: Fine-needle aspiration of the thyroid and correlation with histopathology in a contemporary series of 240 patients. Am J Surg. 2003; 186(6): 702–9; discussion 709–10. PubMed Abstract \| Publisher Full Text\n\nJonklaas J: Importance of Thyroid Hormone Replacement Therapy in Patients with Medullary Thyroid Cancer. In: Medullary Thyroid Cancer. edn.: Springer. 2016: 137–149. Publisher Full Text\n\nBaloch ZW, Cibas ES, Clark DP, et al.: The National Cancer Institute Thyroid fine needle aspiration state of the science conference: a summation. Cytojournal. 2008; 5: 6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCibas ES, Ali SZ; NCI Thyroid FNA State of the Science Conference: The Bethesda System For Reporting Thyroid Cytopathology. Am J Clin Pathol. 2009; 132(5): 658–665. PubMed Abstract \| Publisher Full Text\n\nTessler FN, Middleton WD, Grant EG: Thyroid Imaging Reporting and Data System (TI-RADS): A User’s Guide. Radiology. 2018; 287(1): 29–36. PubMed Abstract \| Publisher Full Text\n\nLiu Q, Djuricin G, Prinz RA: Total thyroidectomy for benign thyroid disease. Surgery. 1998; 123(1): 2–7. PubMed Abstract \| Publisher Full Text\n\nHahn LD, Kunder CA, Chen MM, et al.: Indolent thyroid cancer: knowns and unknowns. Cancers Head Neck. 2017; 2(1): 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCastro MR, Gharib H: Continuing controversies in the management of thyroid nodules. Ann Intern Med. 2005; 142(11): 926–931. PubMed Abstract \| Publisher Full Text\n\nOrell S, Sterrett G, Walters M, et al.: Fine needle aspiration cytology of the thyroid gland. Fine needle aspiration cytology. 2005; 5: 139–142.\n\nRojeski MT, Gharib H: Nodular thyroid disease. Evaluation and management. N Engl J Med. 1985; 313(7): 428–436. PubMed Abstract \| Publisher Full Text\n\nKini S, Miller J, Hamburger J: Cytopathology of thyroid nodules. Henry Ford Hosp Med J. 1982; 30(1): 17–24. PubMed Abstract\n\nYeh MW, Demircan O, Ituarte P, et al.: False-negative fine-needle aspiration cytology results delay treatment and adversely affect outcome in patients with thyroid carcinoma. Thyroid. 2004; 14(3): 207–215. PubMed Abstract \| Publisher Full Text\n\nFareed MM, Al Amro A, Bayoumi Y, et al.: One patient--three head and neck primaries: nasopharyngeal, tongue and thyroid cancers. BMC Res Notes. 2013; 6(1): 432. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKihara M, Hirokawa M, Kudo T, et al.: Calcitonin measurement in fine-needle aspirate washout fluid by electrochemiluminescence immunoassay for thyroid tumors. Thyroid Res. 2018; 11(1): 15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmerican Thyroid Association (ATA) Guidelines Taskforce on Thyroid Nodules and Differentiated Thyroid CancerCooper DS, Doherty GM, et al.: Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid. 2009; 19(11): 1167–1214. [Errata in Thyroid. 2010; 20: 674– 675 and Thyroid. 2010; 20: 942]. PubMed Abstract \| Publisher Full Text\n\nSafneck JR, Kutryk E, Chrobak A, et al.: Fixation techniques for fine needle aspiration biopsy smears prepared off site. Acta Cytol. 2001; 45(3): 365–371. PubMed Abstract \| Publisher Full Text\n\nSuen K: Guidelines of The Papanicolaou Society of Cytopathology for the examination of fine-needle aspiration specimens from thyroid nodules. The Papanicolaou Society of Cytopathology Task Force on Standards of Practice. Diagn Cytopathol. 1996; 15(1): 84–89. PubMed Abstract \| Publisher Full Text\n\nBhambhani S, Kashyap V, Das DK: Nuclear grooves. Valuable diagnostic feature in May-Grünwald-Giemsa-stained fine needle aspirates of papillary carcinoma of the thyroid. Acta Cytol. 1990; 34(6): 809–812. PubMed Abstract\n\nCibas ES, Ali SZ: The Bethesda System for Reporting Thyroid Cytopathology. Thyroid. 2009; 19(11): 1159–1165. PubMed Abstract \| Publisher Full Text\n\nSiddig E: \"Cytomorphological Patterns of Thyroid Lesions among 1646 Sudanese Patients: What we can learn from Fine Needle Aspiration Cytology Retrospective Analysis?\". Harvard Dataverse, V2. 2019. http://www.doi.org/10.7910/DVN/AYEZVP\n\nAbi-Raad R, Prasad M, Baldassari R, et al.: The Value of Negative Diagnosis in Thyroid Fine-Needle Aspiration: a Retrospective Study with Histologic Follow-Up. Endocr Pathol. 2018; 29(3): 269–275. PubMed Abstract \| Publisher Full Text\n\nLucci J: Thyroid FNA Gross Description and Diagnosis Correlation. J Am Soc Cytopathol. 2018; 7(5): S22–S23. Publisher Full Text\n\nChung SR, Suh CH, Baek JH, et al.: The role of core needle biopsy in the diagnosis of initially detected thyroid nodules: a systematic review and meta-analysis. Eur Radiol. 2018; 28(11): 4909–4918. PubMed Abstract \| Publisher Full Text\n\nSyrenicz A, Koziołek M, Ciechanowicz A, et al.: New insights into the diagnosis of nodular goiter. Thyroid Res. 2014; 7(1): 6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCurado MP, Edwards B, Shin HR, et al.: Cancer incidence in five continents, Volume IX. International Agency for Research on Cancer. 2007. IARC Press. Reference Source\n\nHamad HM: Cancer initiatives in Sudan. Ann Oncol. 2006; 17(suppl 8): viii32–viii36. PubMed Abstract \| Publisher Full Text\n\nHowlader N, Noone A, Krapcho M, et al.: SEER Cancer Statistics Review, 1975-2013. National Cancer Institute. Bethesda, MD. 2016. Reference Source\n\nAlseddeeqi E, Baharoon R, Mohamed R, et al.: Thyroid malignancy among patients with thyroid nodules in the United Arab Emirates: a five-year retrospective tertiary Centre analysis. Thyroid Res. 2018; 11(1): 17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLal S, Clare SE, Halas NJ: Nanoshell-enabled photothermal cancer therapy: impending clinical impact. Acc Chem Res. 2008; 41(12): 1842–1851. PubMed Abstract \| Publisher Full Text\n\nPrinz RJ, Sanders MR, Shapiro CJ, et al.: Population-based prevention of child maltreatment: the U.S. Triple p system population trial. Prev Sci. 2009; 10(1): 1–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCaruso D, Mazzaferri EL: Fine needle aspiration biopsy in the management of thyroid nodules. Endocrinologist. 1991; 1(3): 194–202. Reference Source\n\nGharib H, Goellner JR, Johnson DA: Fine-needle aspiration cytology of the thyroid. A 12-year experience with 11,000 biopsies. Clin Lab Med. 1993; 13(3): 699–709. PubMed Abstract \| Publisher Full Text\n\nHa EJ, Baek JH, Lee JH, et al.: Sonographically suspicious thyroid nodules with initially benign cytologic results: the role of a core needle biopsy. Thyroid. 2013; 23(6): 703–708. PubMed Abstract \| Publisher Full Text\n\nLacout A, Chevenet C, Marcy PY: Mummified Thyroid Syndrome. AJR Am J Roentgenol. 2016; 206(4): 837–845. PubMed Abstract \| Publisher Full Text\n\nHahn LD, Kunder CA, Chen MM, et al.: Indolent thyroid cancer: knowns and unknowns. Cancers Head Neck. 2017; 2(1): 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaugen BR: 2015 American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer: What is new and what has changed? Cancer. 2017; 123(3): 372–381. PubMed Abstract \| Publisher Full Text\n\nZimmermann MB, Galetti V: Iodine intake as a risk factor for thyroid cancer: a comprehensive review of animal and human studies. Thyroid Res. 2015; 8(1): 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEltom M, Salih MA, Boström H, et al.: Differences in aetiology and thyroid function in endemic goitre between rural and urban areas of the Darfur region of the Sudan. Acta endocrinol (Copenh). 1985; 108(3): 356–360. PubMed Abstract \| Publisher Full Text\n\nOsman AK: Bulrush millet (Pennisetum typhoides) a contributory factor to the endemicity of goitre in Western Sudan. Ecol Food Nutr. 1981; 11(2): 121–128. Publisher Full Text\n\nNarine N, Thiryayi SA, Perera DM: Fine-needle aspiration cytology of renal clear cell carcinoma metastatic to the thyroid gland. Diagn Cytopathol. 2013; 41(9): 843–845. PubMed Abstract \| Publisher Full Text\n\nLataifeh I, Abdel-Hadi M, Morcos B, et al.: Papillary thyroid carcinoma arising from mature cystic teratoma of the ovary. J Obstet Gynaecol. 2010; 30(8): 884–886. PubMed Abstract \| Publisher Full Text\n\nOtani H, Notsu M, Koike S, et al.: Acute suppurative thyroiditis caused by thyroid papillary carcinoma in the right thyroid lobe of a healthy woman. Thyroid Res. 2018; 11(1): 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhatia KS, Rasalkar DP, Lee YP, et al.: Cystic change in thyroid nodules: a confounding factor for real-time qualitative thyroid ultrasound elastography. Clinical Radiol. 2011; 66(9): 799–807. PubMed Abstract \| Publisher Full Text\n\nGayathri B, Rao KS: Pancytopenia: a clinico hematological study. J Lab physicians. 2011; 3(1): 15–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSood N, Nigam JS: Correlation of fine needle aspiration cytology findings with thyroid function test in cases of lymphocytic thyroiditis. J Thyroid Res. 2014; 2014: 430510. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSyrenicz A: Hashimoto’s disease - from theory to practice. In: Thyroid Res. BioMed Central; 2013; 6: A60. Publisher Full Text\n\nMiller KD, Siegel RL, Lin CC, et al.: Cancer treatment and survivorship statistics, 2016. CA Cancer J Clin. 2016; 66(4): 271–289. PubMed Abstract \| Publisher Full Text\n\nAlevizaki M: Management of hyperparathyroidism (PHP) in MEN2 syndromes in Europe. Thyroid Res. 2013; 6 Suppl 1: S10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGharib H: Fine-needle aspiration biopsy of thyroid nodules: advantages, limitations, and effect. In: Mayo Clin Proc. Elsevier; 1994; 69(1): 44–49. PubMed Abstract \| Publisher Full Text\n\nCastro MR, Gharib H: Thyroid fine-needle aspiration biopsy: progress, practice, and pitfalls. Endocr Pract. 2003; 9(2): 128–136. PubMed Abstract \| Publisher Full Text\n\nMohammed ME, Hassan AM, Abdelhadi HA, et al.: Burden and pattern of cancer in the Sudan, 2000-2006. Br J Med Med Res. 2014; 4(5): 1231–1243. Publisher Full Text\n\nNakao T, Nishikawa M, Hisakado M, et al.: Characteristics and natural course of hypoechoic thyroid lesions diagnosed as possible thyroid lymphomas by fine needle aspiration cytology. Thyroid Res. 2018; 11(1): 8. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "58708", "date": "03 Feb 2020", "name": "Eman Aljufairi", "expertise": [ "Reviewer Expertise Anatomic and cellular pathology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes the results of large number of thyroid FNA in Sudan where similar studies are scarce. Therefore, this study is useful to give us insight about that area. However, it's not clear to me how the FNA categories were made. The authors talk in the beginning about the use of Bethesda system; which is a 6-tier system (unsatisfactory, benign, atypia of uncertain significance, follicular/Hurthle cell lesions, suspicious and malignant). Then, authors divide their results into 4-tiers only excluding both atypical categories (AUS and susp.). Does that mean they always have a definite diagnosis in cytology? Or these are final results correlating with other parameters in these cases?\nIt is also a well known face that cytology can not discriminate in follicular or Hurthle cell lesions between adenoma and carcinoma. Yet in this article, we see some cases labelled as adenoma or carcinoma, while others are labelled as neoplasm. It's not clear how that distinguishing was made. Is it based on availability of histology in some cases? Or other criteria e.g. lymph node metastasis by radiology?\nIt's also slightly unusual as authors stated that anaplastic carcinoma carcinoma is the most common type in the malignant category. Are there any possible causes? E.g. population selection or criteria used?\nAs authors stated lack of follow-up data or surgical excision data for negative cases is a limitation of this study. However, it was not clear if they have any excision data in other categories. If this data is available, I think that will add to the strength of the study and its reflection of the usefulness of thyroid FNA in making diagnosis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "70395", "date": "17 Sep 2020", "name": "Weiwei Zhan", "expertise": [ "Reviewer Expertise ultrasound diagnosis and ultrasound guided intervention of thyroid diseases" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study illustrated the FNA works in Thyroid Nodule Center at the Total Lab Care Clinic in Khartoum state, Sudan, which might represent prevalence of thyroid diseases in Sudanese patients. The information in the research was useful and actual. I approved the article to be indexed with reservations.\nThere were two main questions that need answers:\nHow were these 1646 swellings be measured in clinical works? Using an ultrasound examination or palpation?\n\nThe author wrote in the 'Result' section, that size of swellings correlated with malignancy. Please let readers know the detail about their relationships.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-23
https://f1000research.com/articles/8-1568/v1	02 Sep 19	{ "type": "Case Report", "title": "Case Report: A case of PAI-1 4G/5G heterozygosity causing Budd-Chiari Syndrome", "authors": [ "Domingos Sousa", "Sergio Antunes Silva", "Catarina Jorge", "Rita Martins Fernandes", "Ana Isabel Rodrigues", "Margarida Viana Coelho", "Joana Filipa Guimarães", "Rui Marques Osorio", "Juvenal Morais", "Elena Ríos", "Armindo Figueiredo", "Sergio Antunes Silva", "Catarina Jorge", "Rita Martins Fernandes", "Ana Isabel Rodrigues", "Margarida Viana Coelho", "Joana Filipa Guimarães", "Rui Marques Osorio", "Juvenal Morais", "Elena Ríos", "Armindo Figueiredo" ], "abstract": "Budd-Chiari syndrome (BCS) is a hepatic venous outﬂow obstruction. A 36-year old Caucasian female was admitted with symptomatic hypoglycaemia. Lab tests revealed mild leucocytosis, thrombocytopenia and hepatic cytolysis. The abdominal ultrasound showed mild hepatomegaly due to hypertrophy of the left and caudate lobes, no blood flow on the right and medium hepatic veins and multiple intra-hepatic collateral vessels. Upper endoscopy showed grade I varicose veins. Further studies ruled out common prothrombotic disorders but identified an inherited thrombophilia: a plasminogen activator inhibitor 1 (PAI-1) 4G/5G heterozygous polymorphism. On presentation, this patient had signs of cirrhosis and secondary portal hypertension from imaging results at the time of diagnosis but no symptoms. Four years after the diagnosis the patient continues asymptomatic, which is very unusual. This patient's outcome will be favourable as long as their cirrhosis is compensated by the collateral vessels' permeability. Our case highlights a new association between primary BCS secondary to a prothrombotic inherit mutation: the PAI-1 4G/5G polymorphism.", "keywords": [ "Budd-chiari Syndrome", "Thrombosis", "Hepatology", "Liver disfunction" ], "content": "Introduction\n\nBudd-Chiari syndrome (BCS) is defined as a hepatic venous outflow tract obstruction -regardless of the level or mechanism of obstruction. It is considered primary when the hepatic venous outflow obstruction originates from an endoluminal venous lesion, and secondary in cases of compression or invasion originating from outside the hepatic veins. It can be acute, subacute or chronic in its presentation1–5\n\nAbout 80–85% of the patients have symptoms at the onset of the disease6. Classic manifestations include abdominal pain, fever, ascites and peripheral edema1–5.\n\nFurthermore, an underlying risk factor for thrombosis is found in up to 87% of BCS patients7,8. Myeloproliferative disorders are responsible for 40-50% of primary BCS cases in the presence of JAK2 V617F mutation. There is also an established association with prothrombotic disorders, acquired or inherited, such as factor V Leiden mutation, G20210A prothrombin gene, deficiency in protein C and S, factor II and antiphospholipid syndrome7,8. A genetic variant of the methylenetetrahydrofolate reductase gene (MTHFR) has also been found to increase the risk of BCS9. Plasminogen Activator Inhibitor-1 (PAI-1) mutation leads to impaired fibrinolysis or hypofibrinolysis increasing the risk of prothrombotic disorders. PAI-1 is a crucial physiological inhibitor of fibrinolysis and regulates fibrinolysis by inhibiting tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA) which results in reduced fibrinolytic capacity. The genetic polymorphism in the promoter region of the PAI-1 gene can manifest as the 4G/4G or 4G/5G polymorphism and has been associated with altered PAI-1 plasma concentrations and activity levels. Prothrombotic disorders predispose to hepatic venous outflow tract obstruction and development of BCS.\n\nThe prognosis of BCS patients varies according to the presence and development of liver failure. Stratification and prognosis of BCS is currently obtained using the Model for End-Stage Liver Disease (MELD) and Child-Pugh scores5.\n\nManagement options include anticoagulation as first line therapy and, when appropriate, transjugular intrahepatic portosystemic shunt (TIPS). A combined strategy can lead to a 5-year survival rate between 80–90%. Patients not responding to the above-mentioned measures should be referred for liver transplantation.\n\nWe report a rare case of polymorphism 4G/5G as a cause of a prothrombotic disorder resulting in BCS.\n\n\nCase report\n\nA 36 year old female Caucasian, farmer, farmer, presented to our Accident and Emergency Department with symptomatic hypoglycemia in March 2014 leading to hospital admission for further study. The patient did not indicate any abdominal pain, constitutional symptoms, gastro-intestinal complaints, alcohol or drug consumption and had no relevant changes on physical examination.\n\nThe patient had a past medical history of high blood pressure, obesity, depression and idiopathic thrombocytopenia and was not taking any oral contraceptive pill.\n\nLaboratory results showed (normal ranges in parentheses): white blood count (WBC) 11.600u/l (4000–10.000), haemoglobin (Hb) 11.5g/dl (12.5–14.5), platelets 69.000u/l,(150.000–400.000) International normalized ratio (INR) 1.2 (0.7–1.1), aspartate aminotransferase (AST) 1305IU (10–34), alanine aminotransferase (ALT) 1123IU (10–55), γ glutamyltransferase (GGT) 167UI (<65) creatinine 1.6mg/dl (0.4–1.0), albumin 3.2 (>4.5), total bilirubin 2.2mg/dL (0.6–1.1), direct bilirubin 1.3mg/dL (0.1–0.3), C-reactive protein 55mg/d (<5). Serological testing for viral hepatitis was negative (hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), anti-hepatitis C virus and hepatitis A virus, Epstein-Barr virus, cytomegalovirus).\n\nAbdominal ultrasound with Doppler revealed mild hepatomegaly due to hypertrophy of the left and caudate lobe; heterogeneity of the liver parenchyma; no blood flow in the right and middle hepatic vein; re-permeabilization of the para-umbilical vein; multiple intra-hepatic collateral vessels; splenomegaly and a small amount of free fluid in the pelvic recesses. Upper endoscopy showed grade I varicose veins.\n\nOn MRI scan, the patient had imaging signs of cirrhosis on a magnetic resonance imaging (MRI) scan [Figure 1 and Figure 2], secondary portal hypertension and caudate lobe hypertrophy which allowed the diagnosis of asymptomatic BCS.\n\nThe liver enzymes normalized 2 weeks after admission without any kind of treatment of the underlying causes.\n\nA complete study of thrombotic risk factors did not identify any of the following anomalies: JAK2 V617F mutation, deficiency in protein C and S, Factor II, factor V Leiden mutation, G20210A prothrombin gene, or MTHFTR (677 and 1298). The patient was found to be heterozygous for the 4G/5G variant of the PAI-1 gene. The patient was negative for lupus anticoagulant, anti-b2-glycoprotein and anticardiolipin-b antibodies. The patient was discharged with oral warfarin (5mg/day) and followed up at the anti-coagulation clinic.\n\nThe patient has been attending gynecology appointments, referring due to menometrorrhagia without significant hemoglobin variation while on warfarin. The patient attends the Hepatology Unit every 3 months, and recent lab results display a mild cholestatic pattern with mild anemia. Abdominal CT scans have not revealed any new findings for the last 4 years.\n\n\nDiscussion\n\nPrimary Budd-Chiari syndrome is a venous outflow obstruction that is associated with at least one inherited or acquired prothrombotic risk factor as the underlying cause of thrombosis. The clinical manifestations can be variable but up to 20% of the patients are asymptomatic6. Our patient has a rare form of asymptomatic Budd-Chiari. The absence of symptoms strongly correlates to the development of a large collateral hepatic vein to balance the pathogenesis of portal hypertension of the BCS1,2. A detailed study for prothrombotic disorders must be performed on every patient diagnosed with BCS as patients usually present with at least one prothrombotic risk factor1,2\n\nFor this reason, a schematic approach was used to rule out known risk factors for thrombosis as described in the current literature2,4,5,7. We extended the investigation of inherited thrombophilias and performed a genetic test of PAI-1 that came back positive for 4G/5G heterozygosity.\n\nTo our knowledge this is the first report in the literature to describe an association between heterozygosity for the 4G/5G variant of the PAI-1 and BCS. Hemostasis is a result of the equilibrium between prothombotic and antithrombotic mechanisms in response to tissue injury. The elevation of PAI-1 is a cause of impaired fibrinolysis leading to increased risk of venous thrombosis10. The polymorphism 4G/5G results from a single deletion/insertion of a guanoside residue in promoter region of PAI-111. Inheritance of both 4G alleles (homozygous 4G/4G) has been associated with elevated PAI-1 levels leading to hypofibrinolysis and increased thrombotic risk12. However, this association is still a matter of debate in the medical literature10. The present case report highlights heterozygous 4G/5G as a cause of increased prothrombotic risk, due to elevated PAI-1 levels which caused an hypofibrinolytic state with formation of blood clots within the hepatic vessels, destruction of liver parenchyma ending in BCS13. Our case supports the possible effect of an inherited prothrombotic mutation causing the PAI-1 4G/5G polymorphism that has rarely been described previously.\n\nThe treatment options for BCS include medical management of the underlying risk factors for thrombosis. Prothrombotic drugs such as oral contraceptives are contraindicated. The presence of myeloproliferative disease should prompt immediate treatment of the underlying haematological disorder.\n\nAs such, due to the high prevalence of underlying thrombophilia, anticoagulants are recommended in all patients regardless of the presence of clinical manifestations1,5. Other therapeutic approaches include decompressing therapies such as recanalization strategies (thrombolytic therapy, stenting and angioplasty) surgical shunting and TIPS and, as a last resort, orthotopic liver transplantation (OLT)1,5.\n\nDue to the rarity of this condition and the lack of clinical trials with BCS most treatment options are based on retrospective studies, case reports and expert opinion. Recent developments in imaging techniques and biomolecular tests have made the discovery of underlying causes possible, optimizing treatment strategies and improving overall survival, which is now up to 5 years after the diagnosis in 90% of the cases2.\n\nOur patient was discharged with a vitamin K antagonist (warfarin) as per the international recommendations by an expert panel consensus on management of BCS anticoagulation therapy until the treatment of the underlying condition is achieved, when it is found14.\n\n\nConclusions\n\nWe describe in detail a very rare case of an inherited thrombophilia secondary to a heterozygous 4G/5G polymorphism of the PAI-1 gene, that has not previously been described as associated with BCS. Hence, we suggest that future investigations on BCS must include genetic tests of PAI-1.\n\nFrequently, BCS patient’s prognosis is determined by the development of liver vessels collaterals that compensate the portal hypertension thereby reducing liver disfunction leading to absent physical symptoms. The combined medical and invasive approach can lead to a 5-year survival rate close to 90%.\n\n\nConsent\n\nWritten informed consent for the publication of this case report and any associated images was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nPlessier A, Valla DC: Budd-Chiari syndrome. Semin Liver Dis. 2008; 28(3): 259–269. PubMed Abstract \| Publisher Full Text\n\nDarwish Murad S, Plessier A, Hernandez-Guerra M, et al.: Etiology, management, and outcome of the Budd-Chiari syndrome. Ann Intern Med. 2009; 151(3): 167–175. PubMed Abstract \| Publisher Full Text\n\nJanssen HL, Garcia-Pagan JC, Elias E, et al.: Budd-Chiari syndrome: a review by an expert panel. J Hepatol. 2003; 38(3): 364–71. PubMed Abstract \| Publisher Full Text\n\nPlessier A: [Budd-Chiari syndrome]. Rev Med Interne. 2013; 34(12): 741–5. PubMed Abstract \| Publisher Full Text\n\nMartens P, Nevens F: Budd-Chiari syndrome. United European Gastroenterol J. 2015; 3(6): 489–500. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHadengue A, Poliquin M, Vilgrain V, et al.: The changing scene of hepatic vein thrombosis: recognition of asymptomatic cases. Gastroenterology. 1994; 106(4): 1042–7. PubMed Abstract \| Publisher Full Text\n\nPrimignani M, Barosi G, Bergamaschi G, et al.: Role of the JAK2 mutation in the diagnosis of chronic myeloproliferative disorders in splanchnic vein thrombosis. Hepatology. 2006; 44(6): 1528–1534. PubMed Abstract \| Publisher Full Text\n\nPatel RK, Lea NC, Heneghan MA, et al.: Prevalence of the activating JAK2 tyrosine kinase mutation V617F in the Budd-Chiari syndrome. Gastroenterology. 2006; 130(7): 2031–8. PubMed Abstract \| Publisher Full Text\n\nLi XM, Wei YF, Hao HL, et al.: Hyperhomocysteinemia and the MTHFR C677T mutation in Budd-Chiari syndrome. Am J Hematol. 2002; 71(1): 11–14. PubMed Abstract \| Publisher Full Text\n\nMeltzer ME, Lisman T, de Groot PG, et al.: Venous thrombosis risk associated with plasma hypofibrinolysis is explained by elevated plasma levels of TAFI and PAI-1. Blood. 2010; 116(1): 113–121. PubMed Abstract \| Publisher Full Text\n\nLoskutoff DJ, Linders M, Keijer J, et al.: Structure of the human plasminogen activator inhibitor 1 gene: nonrandom distribution of introns. Biochemistry. 1987; 26(13): 3763–3768. PubMed Abstract \| Publisher Full Text\n\nDawson S, Hamsten A, Wiman B, et al.: Genetic variation at the plasminogen activator inhibitor-1 locus is associated with altered levels of plasma plasminogen activator inhibitor-1 activity. Arterioscler Thromb. 1991; 11(1): 183–190. PubMed Abstract \| Publisher Full Text\n\nSeguí R, Estellés A, Mira Y, et al.: PAI-1 promoter 4G/5G genotype as an additional risk factor for venous thrombosis in subjects with genetic thrombophilic defects. Br J Haematol. 2000; 111(1): 122–8. PubMed Abstract\n\nde Franchis R: Evolving consensus in portal hypertension. Report of the Baveno IV consensus workshop on methodology of diagnosis and therapy in portal hypertension. J Hepatol. 2005; 43(1): 167–176. PubMed Abstract \| Publisher Full Text" }	[ { "id": "54039", "date": "07 Oct 2019", "name": "Cengiz Korkmaz", "expertise": [ "Reviewer Expertise Rheumatology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors reported a case who developed BCS due to heterozygous mutation for PAI-1 gene.The case is interesting and instructive. But I have some concerns:\nIn the past medical history, authors reported that the pt had ITP. What was the last situation on ITH in terms of therapy. Was she under the any treatment such as glucocorticoids?\n\nAuthors must state laboratory parameters correctly according to international rules; anti-b2-glicoprotein? anticardiolipin-b?\n\nIn discussion section, authors should give us a little bit more literature data about the role of PAI-1 gene mutation on deep venous thrombosis.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "5132", "date": "16 Jan 2020", "name": "Domingos Sousa", "role": "Author Response", "response": "Dear reviewer,Many thanks for all the inputsThe manuscript has been reviewed taking your considerations into account1. Under no treatment for ITP. No steroid therapy. 2. anti-b2-glycoprotein (IgM 0.61 UA/mL and IgG <0.10 UA/mL) and anticardiolipin-b antibodies (IgM 2.72 MPL/mL and IgG 3.72 GPL/mL).3. We found new evidence supporting the role of PAI-1 on DVT. Kind regards,Domingos" }, { "c_id": "5164", "date": "17 Jan 2020", "name": "cengiz korkmaz", "role": "Reviewer Response", "response": "Authors must use international writing rule for laboratory results. Please use \"anti-Beta2 GPI antibody\" \"anticardiolipin antibodies (ACA Ig G and M\"" } ] }, { "id": "56811", "date": "25 Nov 2019", "name": "Xingshun Qi", "expertise": [ "Reviewer Expertise Budd Chiari syndrome and portal vein thrombosis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn Introduction section, the authors said \"Myeloproliferative disorders are responsible for 40-50% of primary BCS cases in the presence of JAK2 V617F mutation.\" These words are not accurate. Please see the reference regarding meta-analysis of prevalence of JAK2 V617F mutation in BCS cases (PMID: 21395632)1. Maybe the authors wanted to express the words \"JAK2 positive MPD is about 40-50% in BCS cases\".\n\nIn Introduction section, the results of a meta-analysis found \"the prothrombin G20210A mutation is associated with PVT, but not Budd-Chiari syndrome.\" (PMID: 24793031)2 When the authors said \"There is also an established association with prothrombotic disorders, acquired or inherited, such as factor V Leiden mutation, G20210A prothrombin gene, ...\", please consider the accuracy of your words.\n\nIn Introduction section, please conside the results of a meta-analysis \"Homozygous MTHFR mutation and hyperhomocysteinemia may be associated with the occurrence of BCS\" (PMID: 24773704)3, when you said \"A genetic variant of the methylenetetrahydrofolate reductase gene (MTHFR) has also been found to increase the risk of BCS\". Please emphasize \"homozygous\".\n\nWhen you said \"Plasminogen Activator Inhibitor-1 (PAI-1) mutation leads to impaired fibrinolysis or hypofibrinolysis increasing the risk of prothrombotic disorders.\", please cite a reference.\n\nIs there any evidence regarding the association of PAI-1 mutation with splanchnic vein thrombosis?\n\nPlease review the accuracy of your words \"... female Caucasian, farmer, farmer, presented to ...\".\n\nGive more images of contrast-enhanced MRI scans to demonstrate the hepatic veins.\n\nPlease give the information regarding oral contraceptives.\n\nAs for chronic BCS, please observe the risk of varices. Please consider endoscopy.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "5133", "date": "16 Jan 2020", "name": "Domingos Sousa", "role": "Author Response", "response": "Dear reviewer,Many thanks for all your inputsThanks for the precious suggested references. Unfortunately we do not have more MRI imaging to illustrate our case reportThe manuscript has been reviewed taking your considerations into account.Kind regards,Domingos Sousa" } ] } ]	1	https://f1000research.com/articles/8-1568
https://f1000research.com/articles/8-81/v1	21 Jan 19	{ "type": "Research Article", "title": "Comparative study of the in vitro phytochemicals and antimicrobial potential of six medicinal plants", "authors": [ "Charles O. Nwonuma", "Tabitha A. Adelani-Akande", "Omorefosa O. Osemwegie", "Abiola F. Olaniran", "Toluwani A. Adeyemo", "Charles O. Nwonuma", "Tabitha A. Adelani-Akande", "Abiola F. Olaniran", "Toluwani A. Adeyemo" ], "abstract": "Background: This study sought to investigate the antimicrobial activity of six plants used in traditional medicine in Africa. Methods: The antimicrobial activity of the six medicinal plant extracts (aqueous and ethanol) were evaluated against Proteus mirabilis (ATCC 21784), Pseudomonas aeruginosa (ATCC27856) were Aspergillus fumigatus using the agar-well diffusion protocol. The activities of these extracts were compared with the positive controls chloramphenicol and griseofulvin. Similarly, the phytochemicals from the extracts were qualitatively assayed and their percentage yield calculated by standard methods. Results: The bacterial organisms used, P. mirabilis and P. aeruginosa, were slightly-to-highly susceptible to aqueous and ethanolic extracts from the various test plants, while A. fumigatus was insensitive to the treatments. The ethanolic extracts of the sampled plants showed superior inhibitory performance on the target bacteria to the aqueous extracts. Aqueous and ethanolic extracts of Aframomum melegueta, Moringa oleifera and Cola nitida showed inhibitory consistency against the target bacteria. Superior inhibitory activity was observed for ethanol extracts of A. melegueta seed and M. oleifera pod against P. mirabilis and P. aeruginosa. Variations in phytochemicals were noticed across solvents and plant parts for all plants. Phenols were detected in the aqueous and ethanolic extracts of C. nitida and Cola acuminate, but relatively appeared denser in extracts of A. melegueta seed and Chrysophyllum albidium fruits. The extracts of C. nitida, C. acuminate and A. melegueta tested positive for the presence of flavonoids, which were undetected in C. albidium and M. oleifera seed and pod extracts. None of the extracts showed the presence of every phytochemical assayed during the study. Conclusions: Extracts of the medicinal plants assessed in this study showed antibacterial potential. Developing new methodologies that preserve the bioactive potency of phyto-extracts for optimal microbicidal activity is promising for development of safe, non-reactive pharmaceuticals.", "keywords": [ "Antimicrobial activities", "Medicinal plants", "Phytochemicals", "Bacteriostatic", "Bacteriocidal" ], "content": "Introduction\n\nThe ethnobotanical use of medicinal plants and/or their derivatives (essential oil, resins and soluble extracts) in Africa dates back to early civilization (Sofowora, 1993). This practice is globally perceived as comparatively cheaper and more widely accessible to most rural or less-privileged economies of the world than modern synthetic drugs (Lawal et al., 2012). Statistics as presented by Fabricant & Farnsworth (2001) in the bulletin of the World Health Organization showed that close to 65% of the world population relied on medicinal plants for their primary healthcare drugs (Eddouks & Ghanimi, 2013). Consequently, it is estimated that about 39% of drugs developed since 1980 have being from natural plants, their derivatives or analogues (Newman & Cragg, 2007; Verpoorte et al. (2010). In addition, Ramawa et al. (2009) and Verpoorte et al., (2010) noted that approximately 25% of the currently used modern drugs are derived from plants, a number largely composed of analgesics (e.g. morphine), cardiotonics, chemotherapeutics and antimalarials (e.g. quinine and artemisinin). In recent decades, there have been growing global concerns on the rising cost of buying synthetic drugs, assessing their toxicological profile, and redressing their periodic side-effects and unstable efficacy (Gupta et al., 2016). These undermine their continuous use in modern healthcare delivery and cause a renaissance of herbal screening as well as the chronopharmacological process (Westh et al., 2004). The clinical and pharmacokinetics sustainability of the efficacy of many synthetic antibiotics, prophylactics and curative drugs is threatened by the growing emergence of multi-drug resistant pathogenic strains and cost of production (Bandow et al., 2003). These concerns caused researchers to shift focus and exploit more intensely natural plants and their allies (ferns, fungi and algae) for safer generic bioequivalents to synthetic drugs or substitutions with stable therapeutic values (Rojas et al., 2003; Savoia, 2012). Synthetic drugs use in animal farming also have implications for the global development of organic foods.\n\nPlant-derived medicine accounts for more than a quarter of today’s pharmacopoeia and over US$3.5 billion in annual export value of pharmaceutical (Eddouks & Ghanimi, 2013). While the global inventory of ethnobotanicals is growing, the catalogue of their bioactive compounds that can improve human health is constantly being updated. Over 12,000 bioactive metabolites (primary and secondary) and pigments of plant origin with a wide range of biological activities as well as therapeutic values were documented. Osemwegie et al. (2014) noted the inadequacy of current data in capturing the global totality of medicinal plants due to either neglect of plants in remote ecozones or bias against other related plant biota. While the prehistoric and historic knowledge of numerous health-beneficial plants in Africa seems threatened in recent times, their use in primary healthcare delivery predates modern medicine (Pasewu et al., 2008). This has facilitated the hybridization of both traditional and modern primary healthcare systems in some continents of the world (Eddouks & Ghanimi, 2013).\n\nAframomum melegueta (Roscoe) K. Schumann (Alligator pepper), Chrysophyllum albidum G. Don (Cherry), Cola nitida (Vent.) Schott and Endl., Cola acuminate (P. Beauv.) Schott and Endl. (Kolanuts), and Moringa oleifera Lam. (Moringa) were listed among over 5,000 species of documented medicinal plants reported by Mahomoodally (2013). These plants, from ethnobotanical surveys, were found to be common in many Nigerian culture and traditional festivities, featuring as masticatory and spiritual materials at traditional marriage, coronation and invocation ceremonies (Anwar et al., 2007; Idu et al., 2007; Pasewu et al., 2008). They are also ethnopharmacologically valuable in maintaining, preventing and improving health, and in treating different forms of illness in many African nations (Duraipandiyan et al., 2006; Idu et al., 2007; Mahomoodally, 2013). While the data on West African medicinal plants is hardly current, reports are inconsistent on the use of these plants as insecticides, antimicrobial, molluscicides and nematocides across cultures (Odugbemi, 2006). Similarly, the therapeutic scope and potency of biological active constituents of plants are to a large extent improved by ecosystem factors, methods of extraction of biometabolites, polarity of extraction solvent(s) used and part of plant assayed (Saini et al., 2016; Shitan, 2016). The emerging human allergic response or side effects from synthetic drugs usage, explosion of multi-drug resistant microorganisms, and global paradigm for more efficient stable biopharmacopoeia have increased toxicological, mechanism of action, ethnobotanical and phytometabolomic investigations of medicinal plants. This present study therefore aims to compare the antimicrobial potential, phytochemical profile and solubility of the phytometabolome of selected medicinal plants in aqueous and ethanol media.\n\n\nMethods\n\nSterile distilled water, ethanol, dilute hydrochloric acid, 0.1% ferric chloride solution, microbiological media, antibiotics, sodium chloride, barium chloride, chloroform, acetic anhydride and H2SO4, NaOH were purchased from Ayo-Sigma (ZSA) Chemicals Ltd in Jos, Plateau State, Nigeria.\n\nPseudomonas aeruginosa (ATCC27856), Proteus mirabilis (ATCC21784) and Aspergillus fumigatus strains were used as target organisms for the study. The cultures of bacterial strains used were obtained from immune-compromised patients of the University Health Centre by personnel of the institution’s laboratory. These were preliminarily observed to develop antimicrobial resistance. These strains were later sub-cultured and stored in the Microbial Bank of the Microbiology Laboratory, Landmark University, OmuAran. Aspergillus fumigatus was obtained from composited plant materials and kept in similar banks as the bacterial strains. Each microbe was cultured in bijou bottles containing agar slants of nutrient agar (LAB-008) and Sabourad Dextrose Agar (LAB-009).\n\nCola acuminate [Pal. De Beauv.] Schott and Endl., Cola nitida (Vent.) Schott and Endl., Aframumum melegueta (Roscoe) K. Schum, Moringa oleifera Lam, and Chrysophyllum albidumn G. Don were randomly collected form rainforests in the southern belt of Nigeria. These medicinal plants that were selected based on existing ethnobotanical data were authenticated using picture books of tropical medicinal plants (Fayaz & Ramachandran, 2015; Mueller & Mechler, 2005). Identification was corroborated by the Curator, University of Ilorin Herbarium in Nigeria.\n\nThe seeds of plants used for this study were rid of their coat, air-dried for 14 days and then pulverized with a stainless-steel electric blender. This was then sieved with a 325-mesh sieve before storage in labeled, air-tight, sterile universal bottles. The Moringa, seeds and pods were also air dried separately for 5 days before milling to a powder while the two species of kola nuts (Cola nitida and Cola acuminate) were also sorted, air-dried for 14 days, pounded with a mortar and pestle, air-dried again for another 4 days and stored in air-tight jars on the laboratory bench. In the same vein, the fruit pulp and the fruit apicarp of Chrysophyllum albidum were manually separated from the seeds, air dried together for 12 days and also stored in air-tight jars.\n\nA total of 80 g of each of the pulverized plant materials was weighed, poured into a labeled 500-ml conical flask and then soaked with 400 ml distilled water prior to each being vortexed for effective extraction using a multipurpose vibrator for 18 h. The preparations were then filtered with a steam-sterilized white handkerchief fixed in a glass filter funnel that drained to labeled 250 ml conical flasks. The residue from each plant extraction was stored in labeled cylindrical covered jar and refrigerated while each filtrate was further processed using a rotary evaporator (Model R-205V) at 55°C until a thick concentrate was obtained. This was later transferred into 250 ml beakers and further concentrated on a Water bath at 50°C until a paste was formed (dry crude extract). The paste was later spatulated into freshly labeled sterile universal bottles and weighed (weight of the dry extracts = weight of the universal bottle containing extract – weight of empty universal bottle).\n\nA similar weight (80 g) of each pulverized plant material was weighed and soaked in 400 ml of 95% ethanol in labeled 500 ml conical flasks prior to being vortexed with a bench-top reciprocal shaker (E5850) for a period of 18 h. The mixture was then filtered and processed using the similar extraction protocol for aqueous extraction.\n\nThe percentage extraction yield is expressed as:\n\nExtraction yield (%) = Weight of dry extract (g) x 100/Weight of sample used for extraction (g)\n\nWhere: weight of dry extract is the actual weight of the extracts and the weight of the sample used for the extraction (g) is the initial weight of the samples measured (80 g).\n\nPhytochemical screening of both the aqueous and ethanol extracts of the different plant materials was done according to Tiwari et al (2011). The concentrates were solubilized with either distilled water or ethanol to assay saponins, phenolics, flavonoids and terpenoides of each plant specimen respectively. A total of 0.10g of each of the plant extracts was weighed in a labeled sterile universal bottle, appropriately solubilized with 2 ml of either distilled water or ethanol and transferred into clean labeled test-tubes. The solutions were heated over a Bunsen flame for 3 min, agitated, filtered with Whatmann filter paper (32 mm), cooled, agitated again continuously for 2 min, left to stand for 10 min and observed for froth. In another protocol, phenols, flavonoids and terpenoids were qualitatively investigated using previously described methods (Harborne, 1998). The intensity of color change was used as indicator, observed and rated mildly intense (+), strongly intense (++), extremely intense (+++). Color intensity was visually judged using an RGB color chart.\n\nA total of 28 g of nutrient agar (NA) and 65 g of Sabouraud dextrose agar (SDA) were each weighed into a sterile conical flask containing 1000 ml of distilled water and mixed vigorously. Each flask was then corked with an absorbent cotton wool stopper after which it was autoclaved for 15 mins at 121°C and later allowed to cool. This was then dispensed to labeled Petri dishes under a laminar flow chamber where they were allowed to solidify. The bottom of each of the plates was marked into 4 quadrants while they were still inverted.\n\nIn this study, the McFarland turbidity standard method, as described by Forbes et al. (2007) was used. The 0.5 McFarland standard, which is equivalent to 1.5 X 108 bacteria/ml was prepared according to standard methods (Zapata & Ramirez-Arcos, 2015). Furthermore, normal saline suspensions of the pure culture of each target bacteria were prepared. Turbidity was comparable to the 0.5 McFarland standards by visual determination. Similarly, a serial dilution of Aspergillus fumigatus suspension was done and the 10-3 diluent was then used.\n\nA concentration of 5 mg/ml of chloramphenicol and griseofulvin was prepared, serving as positive controls for antibacterial and antifungal activities, respectively. A solution of 0.85% NaCl was prepared and used as negative control.\n\nThe agar well diffusion method was used for the antimicrobial assay (Murray et al., 2016; Ncube et al., 2008). Previously prepared nutrient agar plates were flooded evenly with 1 ml 1.5 X 108 bacteria/ml of each bacterial strain in triplicate. These were left for 15 min after which four 6 mm wells were bored aseptically with a sterile cork-borer into each inoculated agar plate. Next, 400 mg from each of the prepared crude extract was dissolved in 1 ml of sterile distilled water or ethanol, as appropriate. A total of 100 μl of each plant extracts was then used to fill 4 equidistant wells. For the controls, 100 μl each of the commercial antimicrobials (positive control) and normal saline (negative control) solutions was used to fill agar wells. The plates were all allowed to incubate at 36°C for 24 h for bacteria and room temperature for 48 h for the fungus. These were then observed for zones of inhibition around the wells. Diameters of zones of inhibition were measured in millimeters with a meter rule.\n\n\nResults\n\nThe ethanol and water used in this study elicited varying degrees of solubility of plant phytochemicals. The water extract had the highest yield (yield range, 2.13–22.88%) of the soluble phytochemicals compared to 95% ethanol (yield range, 1.76–22.74%) (Table 1). A marked contrast was observed between the aqueous and ethanol extract yields of Chrysophyllum albidum and Cola nitida respectively. Ethanol had the least phytochemical yield value of 1.76% for Moringa oleifera pod. This was apparently lower than the lowest yield value for the aqueous (2.13%) extract of the same plant material (Table 2).\n\nAssessment of the phytochemical profile of each plant extract showed a marked variation in the phytochemical content of each plant material used in this study. Phenol was detected in all the plant materials evaluated, with the exception of Moringa oleifera seeds and pods. Saponin, flavonoids and terpenoids showed inconsistent distributions across the various plant extracts studied, with Moringa oleifera seeds having the highest terpenoid concentration in aqueous extracts compared to the visual color density of its pod extracts (Table 3). Conversely, Aframomum melegueta was negative for terpenoids in the two extractants used for this study. Levels of saponin were observed to be low but present in Cola nitida and moringa seeds of both aqueous and ethanol extractants respectively (Table 4).\n\n- ,absent; +,present in low amounts; ++,present in medium amounts; +++,present in high amounts.\n\n-, absent; +, present in low amount; ++,present in medium amounts; +++ present in high amounts.\n\nThe plant extracts investigated showed various degrees of antimicrobial activity against the target bacterial organisms. The observed zone of inhibition from the edge of each well differed from one target organism to the other. Raw data for zones of inhibition are available on OSF (Nwonuma, 2019). All the plant extracts assayed showed mild inhibition activity compared to the positive control (Chloramphenicol), to which the target bacteria were radically susceptible to (Figure 1). The negative control wells, containing normal saline solutions, showed no inhibitory activity. Plant extracts derived using ethanol solvents showed better inhibitory capacity than the aqueous extracts against Pseudomonas aeruginosa. In addition, Pseudomonas aeruginosa was the most susceptible target bacterium to the plant extracts used for the study. A susceptibility contrast was, however, observed for Proteus mirabilis treated with the aqueous extracts of Cola nitida. Similarly, the aqueous extracts of Chrysophyllum albidium at 400 mg in 1 ml of distilled water had the best inhibition activity against Pseudomonas aeruginosa and Proteus mirabilis (11.3 mm and 9.4 mm) respectively. The highest inhibitory activities were observed for the ethanol extracts of Cola nitida (11.8 mm), Aframomum melegueta (15.8 mm) and moringa pod (13.4mm) against Pseudomonas aeruginosa. The susceptibility results obtained for both aqueous and ethanol assays of investigated plants on Proteus mirabilis vary and were consistently low.\n\nPlants aqueous extracts inhibition performance on Pseudomonas aeruginosa (A) and Proteus mirabilis (C). Ethanol extract of plants inhibitory performance against Pseudomonas aeruginosa (B) and Proteus mirabilis (D).\n\nAspergillus fumigatus showed no visible susceptibility to the various plant extracts investigated with no clear inhibition zone.\n\n\nDiscussion\n\nHerbal practice is now assuming a global dimension, attracting interest primarily in the standardization of the production chain, quality control and indigenous knowledge inventorying (Venkatasubramanian et al., 2018). The use of herbal knowledge in the development of pharmaceutical industries and primary healthcare in many nations of the world is rapidly growing amidst rising clinical health and unstable therapeutic concerns associated with synthetic drugs. Pharmacopeias derived from natural herbs is now rife in the global pharmaceutical market and has become a huge investment (Mahomoodally, 2013). Furthermore, all the plants selected for this study were indigenous, with ethnomedicinal implications at lower minimum inhibitory concentrations (MICs); they elicit pharmacological, biological or toxicological effects on humans (Ramawa et al., 2009). Conversely, their administrations in healthcare practice in this part of the world remained poorly standardized and without clear therapy specificity. Although every one of the plant materials used for this study are misconceived as having multifunctional bioactive compounds for curing numerous ailments (cancer, fever, infections, inflammations, hypertension, diabetics, obesity, dementia, etc.) and zero side effect in humans, they showed varying level of antimicrobial potential against two prevalent clinical Gram-negative target bacteria.\n\nThe tested microorganisms were observed to be most susceptible to the fruit, seed and pod extracts (ethanol and aqueous) of Cola nitida, Aframomum melegueta and Moringa oleifera respectively. This comparative variation maybe attributed to a number of factors involving the chronopharmacology, storage concentration and separation of electric charges (polarity) of the solvent used and composition of bioactive metabolites in the extracts (Wikaningtyas & Sukandar, 2015). The structural and induced resistance of the target microbes coupled with the diversity and richness of relevant solvated metabolites could have equally accounted for the observed variations of inhibitory capacity in the medicinal plants investigated. The choice of ethanol and water for this study is consistent with several other antimicrobial studies involving medicinal plants that used distilled water or ethanol as preliminary extractants of bioactive metabolites of (Ezeifeka et al., 2004). The aqueous extractant was observed to have solvated more constituents as supported by the percentage yield results across the investigated plant materials than ethanol. This finding is inconsistent with the mean zones of inhibition results obtained for the ethanol extract, which suggests better antimicrobial activity by ethanol derived plant extracts (Ahmad et al., 1998; Abu-Shanab et al., 2004; Bacon et al., 2017; Cowan, 1999; Mothana, et al., 2010).\n\nWhile the mechanism underlying the solubility preference of secondary metabolites in aqueous or ethanol is not fully understood, it is logical to assume that the aqueous extract selectively solvated more of the non-cytotoxic or biologically weak secondary metabolites contrary to the ethanol (95%) that extracted lower but optimized consortium of potent biologically active phytochemicals (Nascimento et al., 2000). It is also philosophical to correlate the superior inhibition action of the ethanol extracts of the plants investigated to the capacity of the extractant to solvate phytochemicals that structurally modulate through reactive functional groups for optimal antimicrobial activity (Vinoth et al., 2012; Wink, 2015). This may therefore suggest that the full representation of phytochemicals assayed in the various plant extracts studied was selective or limited and accounted for the huge variations in the mean zones of inhibition obtained for the experimental extracts and positive control (chloramphenicol) tested. Phenols was detected in all the plant extracts except Moringa oleifera. This may have accounted for the observed antimicrobial capacity of tested extracts and phytotherapeutic popularity the medicinal plants screened (Bukar et al., 2010; Manisha & Vibsha, 2004). The cellular proteins disruption potential and interspecific interactions of phenols or other phytochemicals (flavonoids, tannins and polyketides) may be assumed to be responsible for multi-therapeutic uses of the investigated plants. This finding is, however, inconsistent with the report of Saini et al. (2016) and Fahal et al. (2018), who both observed higher deposits of flavonoids than phenols in the plant seeds and pods. Difference in the extraction and susceptibility test protocols may have accounted for the contradiction in experimental observations and the detection of more of the assayed phytochemicals in the fruit extracts of Colanitida.\n\nAlthough this study showed that Pseudomonas aeruginosa is relatively more sensitive to both crude aqueous and ethanol-based plant extracts than Proteus mirabilis, the mechanism underlying the sensitivity reaction remains unclear. In theory, the plant extracts are made up of biochemicals with specific and non-specific modes of actions that compromise the resistance of the target bacteria. The potency and efficacy of the extracts are measured by collective modes of action of the phytochemicals and the corresponding reaction(s) of the target organisms (structural and inductive resistance). Mild resistance posed by Proteus mirabilis could have induced by chemical signaling mechanism (Venkatasubramanian et al., 2018). Further study on the isolation and identification of the phytochemical agent(s) responsible for the observed antimicrobial activity using modern techniques involving High Performance Liquid Chromatography and gas chromatography may be required for better understanding of the mode of action. The inhibition of growth of target bacteria is also attributed to the destruction of cell protein machinery such as ion channels and pumps, enzyme actions, cytoskeleton function and membrane biochemical modulation (Ekpendu, 1995; Idowu et al., 2006; Wink, 2015). The structural complexity of the test fungus Aspergillus fumigatus may have been responsible for its non-susceptibility to all the investigated extracts compared to the positive control of commercial griseofulvin.\n\nThis preliminary study confirmed all the plants investigated as having antibacterial property and corroborating their prevalent use in Nigeria herbal healthcare services. Similarly, ethanol as an extraction solvent optimized more effectively the antimicrobial agents released in the plant extracts compared to the aqueous solvent, supporting the common practice as well as offering the scientific basis to employing tincture for most local herbal preparations. The therapeutic usage of Moringa oleifera leaf however predominates over other plant parts (e.g. roots, barks, pods and fruits) in this part of the world in herbal healthcare predisposing it to possible exploitation for pharmacopeia. It was observed from this study that its seeds had phytochemical efflux with low antimicrobial activity relative to the other plants screened. While Chrysophyllum albidum was the least ethnobotanically popular of the plant studied (Adewoye et al., 2011; Idu et al., 2007; Okoli & Okere, 2010), its fruit extracts proved to have antimicrobial activity that is comparable with the fruits of kola nuts and the alligator pepper. Qualitative assays for flavonoids, terpenoides, phenolics and saponin in all the extracts showed the prevalence of phenolics and inconsistent distribution of the other phytochemicals. Consequently, this by no means accounted for the total phytochemical representations of the plant extracts studied due to non-detection of some unsolvated ones but proved that they have the potential for use as safe, cheap and alternative sources of antimicrobials, and other pharmaceuticals (Abdul et al., 2010; Abu-Shanab et al., 2004).\n\n\nData availability\n\nComplete raw data containing the zones of inhibition for each extract are available on OSF. DOI: https://doi.org/10.17605/OSF.IO/5APVE (Nwonuma, 2019).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAbdul M, Sarker AA, Islam S, et al.: Cytotoxic and antimicrobial activity of the crude extract of Abutilon indicum. International Journal of Pharmacognosy and Phytochemical Research. 2010; 2(1): 1–4. Reference Source\n\nAbu-Shanab B, Adwan G, Abu-Safiya D, et al.: Antibacterial activities of some plant extracts utilized in popular medicine in Palestine. Turk J Biol. 2004; 28: 99–102. Reference Source\n\nAdewoye EO, Adewoye EO, Salami AT, et al.: The Antimicrobial and kill kinetics of Chrysophyllum albidum stem bark extracts. Eur J Sci Res. 2011; 56(3): 434–444. Reference Source\n\nAhmad I, Mehmood Z, Mohammad F: Screening of some Indian medicinal plants for their antimicrobial properties. J Ethnopharmacol. 1998; 62(2): 183–193. PubMed Abstract \| Publisher Full Text\n\nAnwar F, Latif S, Ashraf M, et al.: Moringa oleifera: a food plant with multiple medicinal uses. Phytother Res. 2007; 21(1): 17–25. PubMed Abstract \| Publisher Full Text\n\nBacon K, Boyer R, Denbow C, et al.: Evaluation of different solvents to extract antibacterial compounds from jalapeño peppers. Food Sci Nutr. 2017; 5(3): 497–503. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBandow JE, Brötz H, Leichert LI, et al.: Proteomic approach to understanding antibiotic action. Antimicrob Agents Chemother. 2003; 47(3): 948–955. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBukar A, Uba A, Oyeyi TI: Antimicrobial profile of Moring aoleifera Lam. extracts against some food-borne microorganisms. Bayero Journal of Pure and Applied Sciences. 2010; 3(1): 43–48. Publisher Full Text\n\nCowan MM: Plant products as antimicrobial agents. Clin Microbiol Rev. 1999; 12(4): 564–582. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDuraipandiyan V, Ayyanar M, Ignacimuthu S: Antimicrobial activity of some ethnomedicinal plants used by Paliyar tribe from Tamil Nadu, India. BMC Complement Altern Med. 2006; 6: 35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEddouks M, Ghanimi D: The use of medicinal plants in human healthcare: a scoop on safety. Pharm Regul Aff. 2013; 3: e122. Publisher Full Text\n\nEkpendu TE: A study of the extractives of 3 medicinal plants. Ph.D Thesis, Chemistry, Sciences, University of Ibadan. 1995.\n\nEzeifeka GO, Orji MU, Mbata TI, et al.: Antimicrobial activity of Cajanas cajan, Garcinia kola and Xylopia aethiopica on pathogenic microorganisms. Biotechnology. 2004; 3(1): 41–43. Publisher Full Text\n\nFabricant DS, Farnsworth NR: The value of plants used in traditional medicine for drug discovery. Environ Health Perspect. 2001; 109 Suppl 1: 69–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFahal ME, Rani BMA, Aklakur MD, et al.: Qualitative and quantitative phytochemical analysis of Moringa oleifera (Lam) Pods. International Journal of Current Microbiology and Applied Sciences. 2018; 7(5): 657–665. Publisher Full Text\n\nFayaz PP, Ramachandran HD: Extraction of phytochemical, screening and antioxidant activity of Garcinia indica choisy fruit. World J Pharm Pharm Sci. 2015; 3(12): 726–731. Reference Source\n\nForbes BA, Sahm DE, Weissfeld AS: Bailey and Scott’s Diagnostic Microbiology. 12thEdn. Mosby Inc.; an affiliate of Elsevier, Inc., USA. 2007. Reference Source\n\nGupta S, Tang C, Tran M, et al.: Effect of Predatory Bacteria on Human Cell Lines. PLoS One. 2016; 11(8): e0161242. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarborne JB: Phytochemical Methods: A Guide To Modern Techniques of Plant Analysis. 2nd Edition, Chapmann and Hall Publishers, London, 1998.\n\nIdowu TO, Iwalewa EO, Aderogba MA, et al.: Antinociceptive, Anti – inflammatory and Antioxidant activities of Eleagnine: an alkaloid isolated from Chrysophyllum albidum Seed Cotyledons. J Biol Sci. 2006; 6(6): 1029–1034. Publisher Full Text\n\nIdu M, Ndukwu BC, Osemwegie OO: Ethnofloristic studies of Ethiope Council Area of Delta State, Nigeria. Journal of Plant Sciences. 2007; 2(1): 1–13. Publisher Full Text\n\nLawal HO, Etatuvie SO, Fawehinmi AB: Ethnomedicinal and pharmacological properties of Morinda lucida. J Nat Prod. 2012; 5: 93–99. Reference Source\n\nMahomoodally MF: Traditional medicines in Africa: an appraisal of ten potent african medicinal plants. Evid Based Complement Alternat Med. 2013; 2013: 617459. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nManisha T, Vibsha K: Medicinal Plants. India Tandon Publishers, 2004; 2. Reference Source\n\nMothana RA, Abdo SA, Hasson S, et al.: Antimicrobial, antioxidant and cytotoxic activities and phytochemical screening of some yemeni medicinal plants. Evid Based Complement Alternat Med. 2010; 7(3): 323–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMueller MS, Mechler E: The garden to companion medicinal plants: an A-Z of healing plants and home remedies. Monique Simmonds, Melanie-Jayne Howes and Jason Irving (eds.). GoergeThieme Verlag. 2005; 217.\n\nMurray P, Rosenthal K, Pfaller M: Medical Microbiology. 8th Edn. Mosby Inc., Elsevier Publishing. 2016; 848. Reference Source\n\nNascimento GF, Locatelli J, Freitas PC, et al.: Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Braz J Microbiol. 2000; 31: 247–256. Publisher Full Text\n\nNcube N, Afolayan SAJ, Okoh AI: Assessment techniques of antimicrobial properties of natural compounds of plant origin: current methods and future trends. Afr J Biotechnol. 2008; 7(12): 1797–1806. Publisher Full Text\n\nNewman DJ, Cragg GM: Natural products as sources of new drugs over the last 25 years. J Nat Prod. 2007; 70(3): 461–477. PubMed Abstract \| Publisher Full Text\n\nNwonuma CO: Antimicrobial Potential of Medicinal Plants. OSF. 2019. http://www.doi.org/10.17605/OSF.IO/5APVE\n\nOdugbemi T: Medicinal plants as antimicrobials. In: outline and pictures of medicinal plants from Nigeria. University of Lagos Press. 2006; 53–64.\n\nOkoli BJ, Okere OS: Antimicrobial activity of the phytochemical constituents of Chrysophyllum albidum Plant. J Res Nat Dev. 2010; 8(1). Reference Source\n\nOsemwegie OO, Okhuoya JA, Dania TA: Ethnomycological conspectus of West African mushrooms: An awareness document. Adv Microbiol. 2014; 4(1): 39–54. Publisher Full Text\n\nPasewu GA, Cutler RR, Humber DP: Antibacterial activity of plants used in traditional medicines of Ghana with particular reference to MRSA. J Ethnopharmacol. 2008; 116(1): 102–111. PubMed Abstract \| Publisher Full Text\n\nRamawa KG, Das S, Mathur M: The chemical diversity of bioactive molecules and therapeutic potential of medicinal plants. In herbal drugs: Ethnomedicine to Modern Medicine. 2009; 7–32. Publisher Full Text\n\nRojas R, Bustamante B, Bauer J, et al.: Antimicrobial activity of selected Peruvian medicinal plants. J Ethnopharmacol. 2003; 88(2–3): 199–204. PubMed Abstract \| Publisher Full Text\n\nSavoia D: Plant-derived antimicrobial compounds: alternatives to antibiotics. Future Microbiol. 2012; 7(8): 979–990. PubMed Abstract \| Publisher Full Text\n\nSaini RK, Sivanesan I, Keum YS: Phytochemicals of Moringa oleifera: a review of their nutritional, therapeutic and industrial significance. 3 Biotech. 2016; 6(2): 203. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShitan M: Secondary metabolites in plants: transport and self-tolerance mechanisms. Biosci Biotechnol Biochem. 2016; 80(7): 1283–1293. PubMed Abstract \| Publisher Full Text\n\nSofowora A: Screening Plants for Bioactive Agents: Medicinal Plants and Traditional Medicine in Africa. 2nd Edition, Spectrum Books Ltd., Sunshine House, Ibadan, Nigeria. 1993; 134–156.\n\nTiwari P, Kumar B, Kaur M, et al.: Phytochemical screening and extraction: a review. International Pharmaceutica Sciencia. 2011; 1(1): 36–106. Reference Source\n\nTrease GE, Evans WC: Pharmacognosy. 15th Edition. WB Saunders, London. 2002; 406. ISBN: 8131200876.\n\nVenkatasubramanian P, Balasubramani SP, Nandi SK, et al.: Bioactive metabolite profiling for identification of elite germplasms: a conservation strategy for threatened medicinal plants. Curr Sci. 2018; 114(3): 554–561. Reference Source\n\nVerpoorte R, Mander L, Lui HW (Eds.): Overview and introduction. In Comprehensive Natural Products-II, Chemistry and Biology.2010; 3: 1–4.\n\nVinoth B, Manivasagaperumal R, Balamurugan S: Phytochemical analysis and antibacterial activity of Moringa oleifera Lam. International Journal of Research in Biological Sciences. 2012; 2(3): 98–102. Reference Source\n\nWesth H, Zinn CS, Rosdahl VT, et al.: An international multicenter study of antimicrobial consumption and resistance in Staphylococcus aureus isolates from 15 hospitals in 14 countries. Microb Drug Resist. 2004; 10(2): 169–176. PubMed Abstract \| Publisher Full Text\n\nWikaningtyas P, Sakandar EY: The antibacterial activity of selected plants towards resistant bacteria isolated from clinical specimens. Asian Pac J Trop Biomed. 2015; 6(1): 16–19. Publisher Full Text\n\nWink M: Modes of action of herbal medicines and plant secondary metabolites. Medicines (Basel). 2015; 2(3): 251–286. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZapata A, Ramirez-Arcos S: A comparative study of McFarland turbidity standards and the Densimat photometer to determine bacterial cell density. Curr Microbiol. 2015; 70(6): 907–9. PubMed Abstract \| Publisher Full Text" }	[ { "id": "43338", "date": "04 Feb 2019", "name": "Armen Trchounian", "expertise": [ "Reviewer Expertise Microbiology", "Microbial Biotechnology", "Plant Biotechnology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is of partial interest, the study is too preliminary and poor and should not be considered for indexing in this journal.\nComments:\nAbstract: Results are not actually summarized. Conclusions can be altered for the reflection of all content of the article. The presented conclusions are incomprehensible. A voucher specimen must be deposited in a recognized herbarium (collection) in case of plants, or otherwise an appropriate chemical fingerprint is required for future reference. There are no proper negative controls. Particularly sterile distilled water and ethanol should be used as negative controls at the concentrations they present in the final test solution. Taking into account that ethanol has antiseptic properties how the authors can distinguish detected antimicrobial activity as result of ethanol or tested phytochemicals action. In figure 1 there are six columns regarding the inhibition zones of positive control chloramphenicol differing each other against the same bacterial strains. This is not clear: why? Most good journals do not accept agar diffusion studies to determine the antimicrobial activity of plants. Many factors influence the agar diffusion assay for plant extracts and results between different laboratories cannot be compared. Agar diffusion assays may work well for single chemical compounds but not for plant extracts containing compounds with different polarities. Non-polar compounds do not diffuse well into the aqueous agar matrix and this underestimates activity. MIC using serial dilution delivers reproducible results to compare results in different laboratories and only extracts with MICs less than 0.1 mg/ml are considered, as interesting ones. Using crude extracts of plant materials with concentrations above 1000 μg/ml in antimicrobial screening protocols should be avoided, because using high concentrations of plant crude extracts can bring to false positive results (Rios and Recio, 2005). During the current study authors used 400 mg/ml concertation of the extracts in antimicrobial tests, which is too high. The concentrations of positive controls are also too high (5 mg/ml) In the article, it is stated that bacterial test strains (Pseudomonas aeruginosa (ATCC27856) and Proteus mirabilis (ATCC21784)) were isolated from immune-compromised patients of the University Health Centre and available in Microbial Bank of the Microbiology Laboratory, Landmark University. But the ATCC reference numbers were given to them. Are they available in ATCC's microorganism collection? The discussion must be completely rearranged. Language is poor.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "4399", "date": "30 May 2019", "name": "Osarenkhoe Osemwegie", "role": "Author Response", "response": "We sincerely thank you for the thorough analytics and comments raised. We have already initiated efforts at addressing the issues of comment and wish to respond to some of the issues we disagreed in your reviewer’s comment. There are no proper negative controls. Particularly sterile distilled water and ethanol should be used as negative controls at the concentrations they present in the final test solution. Taking into account that ethanol has antiseptic properties how the authors can distinguish detected antimicrobial activity as result of ethanol or tested phytochemicals action. The method adopted only used ethanol as an extractant of bioactive phytochemicals of the test medicinal plants and not meant for solubilizing the crude for antimicrobial assay. We regret such error and appreciate your pointing it out. Similarly, the extracts were only suspended as crude in distilled water. Therefore, the antiseptic reference of ethanol is not appropriate. In figure 1 there are six columns regarding the inhibition zones of positive control chloramphenicol differing each other against the same bacterial strains. This is not clear: why? The method only affords one positive and one negative control per treatment. No inhibition was observed and recorded (suppressed column) for the negative control treatments hence the number of columns presented. Differing reaction by the same strain of bacterium to in vitro antibiotic treatment is a possibility possible linked to undetectable slow differing changes in the local micro-environmental conditions. Most good journals do not accept agar diffusion studies to determine the antimicrobial activity of plants. Many factors influence the agar diffusion assay for plant extracts and results between different laboratories cannot be compared. Agar diffusion assays may work well for single chemical compounds but not for plant extracts containing compounds with different polarities. Non-polar compounds do not diffuse well into the aqueous agar matrix and this underestimates activity. MIC using serial dilution delivers reproducible results to compare results in different laboratories and only extracts with MICs less than 0.1 mg/ml are considered, as interesting ones. Using crude extracts of plant materials with concentrations above 1000 μg/ml in antimicrobial screening protocols should be avoided, because using high concentrations of plant crude extracts can bring to false positive results (Rios and Recio, 2005). During the current study authors used 400 mg/ml concertation of the extracts in antimicrobial tests, which is too high. The concentrations of positive controls are also too high (5 mg/ml). One 2019 (Arunkumar et al., 2019) reference has been used to reflect currency and spread of literature review. On the technical dept. of the methods, the preference for disc technique in susceptibility test expressed by the version 1 report is scientifically debatable and appreciated. Well diffusion method is also listed among the approved routine antimicrobial susceptibility testing methods in clinical laboratory practices. Selection of method is influenced by a range of factors that may include the nature of culture media, test material and target organism. Literature as recent as 2019 also exists that have employed this method (Mounyr et al., 2016 – Journal of Pharmaceutical Analysis; Arunkumar et al., 2019 – Molecules). Ethanol used on crude was 0.1% while the concentration of crude extract is 200mg/ml instead of 400mg/ml. The three (3) reviewers called fungal spore suspension concentration (105 spores/ml) which is provided in the Assessment of antimicrobial activity). We partially concur with you that well diffusion method is becoming antiquated but its ineffectiveness in investigating the antimicrobial property of plant extracts remains debatable and it is still receiving acceptance in reputable journal outlets (Balouiri et al., 2016 – http://dx.doi.org/10.1016/j.jpha.2015.11.005). We wish for you to know that the first intent of the study was to prove that all the test plants have antimicrobial activity. We refer you to the last paragraph of the discussion which clearly mentioned that the work is preliminary. The concentration used was set based on the outcome of ethnobotanical survey of the location of study and it still generated the expected result despite disagreeing with Rios and Recio, 2005.All other comments on the voucher number for the test plants, reconstruction of the abstract and the discussion, expressive narration of the results will be addressed. While we do not understand what you mean by the language is poor, we wish to appeal for your guidance in the use of the right language hence we would be glad you return to use the edited version of the article.Thank you." } ] }, { "id": "43814", "date": "19 Mar 2019", "name": "Bhim Pratap Singh", "expertise": [ "Reviewer Expertise Microbial secondary metabolites", "traditional medicinal plants", "DNA fingerprinting" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work done by Nwonuma et al. is a interesting work but lacking proper execution. I felt that authors should have done the assays at-least till quantitative analysis.\n\nMajor issue is the use of strain ATCC 21784, which shows as Rhodococcus sp. (21784) at ATCC site (https://www.google.com/search?q=ATCC+21784&rlz=1C1RUCY_enIN705IN705&oq=ATCC+21784&aqs=chrome..69i57.549j0j7&sourceid=chrome&ie=UTF-8) Were Aspergillus fumigatus using, bad language, language of the manuscript needs revision. Aspergillus fumigatus, accession number?\n\nI would suggest a serious revision of the manuscript before accepting for indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "45881", "date": "17 Apr 2019", "name": "Patchima Sithisarn", "expertise": [ "Reviewer Expertise Antimicrobial", "antiviral targeting in pathogenic zoonotic organisms from phytochemicals." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe concept of the work is very interesting about the antimicrobial activities of medicinal plants in Africa. Though there are some crucial points needed to be clarified scientifically.\n\nIn the title, declaration of 6 plants, though there are 5 plants. It is needed to clarify that they are different preparations of extracts not different species. It is crucial scientifically to describe the methods e.g. quantification of A. fumigatus and its experimental standardization as it is fungi. How the fungi grows and be inhibited by extracts in agar which should be different from other organisms which are bacteria that should not be measure in exact same way. Crucial statistical analysis and validity of the data is required to compare antimicrobial activities of the plants to antibiotic of choices.\n\nPhytochemical analysis in the research is qualitative. It may not be used as a referable or validate results in a quality scientific paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/8-81
https://f1000research.com/articles/8-229/v1	28 Feb 19	{ "type": "Research Article", "title": "Characteristics of successful integrated family planning and maternal and child health services: Findings from a mixed-method, descriptive evaluation", "authors": [ "Anne Pfitzer", "Christina Maly", "Hannah Tappis", "Mark Kabue", "Devon Mackenzie", "Sadie Healy", "Vineet Srivastava", "Gathari Ndirangu", "Christina Maly", "Hannah Tappis", "Mark Kabue", "Devon Mackenzie", "Sadie Healy", "Vineet Srivastava", "Gathari Ndirangu" ], "abstract": "Background: Most postpartum women in low- and middle-income countries want to delay or avoid future pregnancies but are not using modern contraception. One promising strategy for increasing the use of postpartum family planning (PPFP) is integration with maternal, newborn and child health (MNCH) services. However, there is limited evidence on effective service integration strategies. We examine facilitators of and barriers to effective PPFP integration in MNCH services in Kenya and India.\n\nMethods: We conducted a cross-sectional, mixed-method study in two counties in Kenya and two states in India. Data collection included surveying 215 MNCH clients and surveying or interviewing 82 health care providers and managers in 15 health facilities across the four sites. We analyzed data from each country separately. First, we analyzed quantitative data to assess the extent to which PPFP was integrated within MNCH services at each facility. Then we analyzed qualitative data and synthesized findings from both data sources to identify characteristics of well and poorly integrated facilities. Results: PPFP integration success varied by service delivery area, health facility, and country. Issues influencing the extent of integration included availability of physical space for PPFP services, health workforce composition and capacity, family planning commodities availability, duration and nature of support provided. Conclusions: Although integration level varied between health facilities, factors enabling and hindering PPFP integration were similar in India and Kenya. Better measures are needed to verify whether services are integrated as prescribed by national policies.", "keywords": [ "family planning", "postpartum", "health services", "integration", "maternal and child health" ], "content": "Introduction\n\nIn low- and middle-income countries, nearly one-fifth of birth intervals are less than 24 months, increasing health risks for both mothers and children1–3. A large proportion of these births likely results from unintended pregnancies among women in the first two years postpartum4. Worldwide, most women want to avoid or delay another pregnancy for a couple of years after having a baby, but many are not using contraception4.\n\nThe promotion of postpartum family planning (PPFP) is a strategy for addressing unmet needs for contraception by helping women to start a chosen contraceptive method soon after birth and either continuing with this method or choosing another to use for at least two years. Increasingly, the integration of PPFP from pregnancy to the extended postpartum period (of two years following childbirth) is seen as a promising strategy for increasing the availability and utilization of PPFP services5–7. Integration, in this context, simply means the deliberate joining together of “inputs, organization and delivery of particular functions” to increase efficiency and access to health services8. Stakeholders and scholars agree that more research is needed on how to effectively integrate PPFP in low- and middle-income countries9. A Cochrane review of primary health service integration found that most studies with specific reference to family planning involved efforts to add family planning to HIV services8. This review found limited evidence that adding family planning to other services leads to greater contraceptive use, and another recent review argued for more evaluation research10. A third systematic review concluded that there was insufficient evidence, particularly about models for implementing PPFP programs10,11. Although there is not conclusive evidence on the efficiency of family planning integration, evidence is mounting on the benefits of using every contact with pregnant and postpartum women to offer FP counseling and services12.\n\nDefinitional issues cloud the integration literature, with some researchers focused on integration of services at the point of care and others focused on integration of national programs to channel resources and technical support for service delivery. When national programs are integrated (or ‘horizontal’), the health system supports integrated services with planning, monitoring, supervision, and at times integrated supply chains, among other provisions13. There is no evidence (yet) if service integration at scale requires program integration. Integrated services are assumed to be more efficient, in particular from the client’s perspective, but also for the health system14. However, analysis of costs show wide variations in service integration between smaller facilities versus larger ones14. From the provider’s perspective, there are concerns of added workload, which are somewhat balanced with increased job satisfaction15.\n\nCapitalizing on programmatic experience from PPFP programs in over 20 countries, we designed a study to assess the integration of PPFP with maternal, newborn, and child health (MNCH) services used by pregnant and postpartum women. Understanding factors affecting success or failure of integrated service delivery in different settings, i.e. increased adoption of multiple services with no decrease in quality, will assist health program managers to plan PPFP programs and focus their service integration efforts where they are most needed.\n\n\nMethods\n\nThis paper presents findings from one component of a descriptive evaluation of PPFP service integration conducted in India (May–June 2014) and Kenya (June-July 2014). We designed this cross-sectional, mixed-methods study16 to examine how PPFP services are provided and identify factors enabling and limiting integration of PPFP with MNCH services in different settings.\n\nWe selected India and Kenya as study sites because of their long-running PPFP programs, which provided us a unique opportunity to evaluate provider and client experiences from different PPFP program models. The study was conducted in two of the 29 states in India (Jharkhand and Bihar), and two of the 47 counties in Kenya. (Embu and Siaya).\n\nPPFP integration programs began in five states in India in 2010 and provided support for introducing PPFP counseling during antenatal care (ANC) and reorganizing intrapartum and immediate postpartum care to offer postpartum intrauterine device (PPIUD) services immediately after birth. This integration of services built upon the rise in institutional deliveries following the Janani Suraksha Yojana (JSY) incentive scheme established in 2005. Jharkhand was among the first five states to initiate PPFP services. Hospital providers received both a 3-day training on clinical services for PPIUD as well as a 2-day training in PPFP counseling. Hospitals also received tools and job aids to support counseling, services and data management, and program staff offered supportive supervisions visits jointly with health authorities. In Bihar, India, starting in 2012, programs have supported the same approach as in Jharkhand but added community outreach and messaging on birth spacing and PPFP during home visits through a 1-day training of Accredited Social Health Activists (ASHAs) and auxiliary nurse midwives on family planning methods and key PPFP messages. Subsequent to experiences in the first states, PPFP continued to expand across India. Furthermore, the government created a dedicated PPFP counselor position and opened an initial 1,300 posts. With the advent of the 2013 reproductive, maternal, newborn and child and adolescent health (RMNCH+A) strategy which incorporated PPFP with intent to make services available across India, the counselors became RMNCH+A counselors.\n\nIn Kenya, PPFP programs began on a small scale in Embu from 2006 to 2010 as part of a pilot study, and then through a Ministry of Health sanctioned training on integrated maternal and newborn and family planning training. This training consisted of an orientation on PPFP for providers in facilities with a high volume of deliveries, followed by a 5-day clinical training in PPIUD and infection prevention for nurse-midwives. Community health workers also received a 2-day orientation on PPFP. Program staff worked with county officials to offer supportive supervision and to identify PPFP “champions” to support postnatal care and PPFP. Meanwhile at national level, the 2009–2015 National Reproductive Health Strategy included the integration of family planning into other reproductive health services as a key approach for improving access to comprehensive reproductive health services. The National Family Planning Guidelines for Service Providers 2010 specifies the postpartum timing for initiating various contraceptive methods. In 2012, a program in Siaya, Kenya, used a multidisciplinary approach to demonstrate integration of family planning and maternal, infant, young child nutrition (FP/MIYCN) into antenatal, intrapartum, postnatal, and early childhood care services at facility- and community-levels. A baseline informed this demonstration project and the development of materials, followed by capacity building of facility providers, community health volunteers on how to counsel on family planning and nutrition, integrate FP/MIYCN in all service delivery areas (maternity, maternal and child health/family planning, outpatient department) and manage program data. Bondo subcounty- (formally referred to as a district) was the only subcounty- out of six in Siaya where PPFP and nutrition services had been introduced and continued through the time of the study. Unlike the other three locations, the Bondo facilities did not offer PPIUD as an option immediately after birth. At the sub-county hospital, FP/MIYCN training took place onsite for three days along with a one-day whole-site orientation, while health center providers underwent five days of training with clinical practice.\n\nIn consultation with local health authorities, we purposively selected 15 health facilities in the two countries: six public health facilities from Bondo subcounty in Kenya and three facilities each from Embu county in Kenya, and Jharkhand and Bihar states in India. In India, the facilities were chosen based on health officials’ assessment of high performance, given the study objective to learn about what works to integrate PPFP. In Embu, Kenya, the health facilities were selected based on both performance and ease of access. There were six public facilities included in Bondo to capture all facility types participating in the nutrition and PPFP integration program. We published a separate manuscript on the family planning and nutrition integration experience from Bondo subcounty17. Data collection methods in this study included a client flow analysis (Dataset 1, published elsewhere18,19); surveys of health care MNCH clients (Dataset 2) and providers (Dataset 3), and in-depth interviews with local authorities, facility management and health care providers. A convenience sampling approach was used for provider surveys and interviews. The number of participants at each facility was determined by the number of PPFP providers expected to be on duty, not a sample size/power calculation or content saturation.\n\nThe study teams obtained letters of support from state- or county-level officials, alerted facilities ahead of arrival, then met with the person in charge for briefing and identification of providers available at the facility. Providers on duty on the day of interviews were approached for interviews or surveys. A study information sheet was posted at each facility and provided to in-charge and anyone else upon request.\n\nMNCH service provider and client surveys were conducted at all 15 health facilities. At each facility, the number of providers surveyed using the structured questionnaire varied between one and four depending on the number of clinical staff at a facility and types of services provided. We collected data on the proportion of consultations that include PPFP counseling as well as their perceptions on PPFP services they provided. With regard to clients, we surveyed a sample of pregnant women and mothers of children under age 2 years attending maternal or child health services to determine whether they received PPFP counseling during their visit on the day of the survey. For the client survey, the number of respondents sampled was designed to provide a study power of 80% to estimate ±10% with 95% confidence, in the estimation of the proportion of clients receiving integrated PPFP services.\n\nData were also collected on wait time before consultation with a service provider, privacy during consultations (audio and visual), comfort discussing private/sensitive health matters, perceptions of service providers’ behavior towards them, and the availability of medications and FP commodities. Surveys were conducted using tablets loaded with CommCare software (CommCare v.2.11.0, Dimagi Inc.); it took 1–3 days to complete the surveys at each health facility. In India, service provider and client surveys were conducted in either Hindi or English depending on a respondent’s preference. In Kenya, all service provider surveys were conducted in English whereas client surveys were conducted in English, Swahili, or Luo, depending on the respondent’s preference. Surveys are available on Open Science Framework20.\n\nClient flow analysis was conducted at two facilities in each Indian state and in three facilities in each county in Kenya; five facilities with low client loads were excluded. Client flow tools were modified from a tool used previously to assess integration of HIV and sexual and reproductive health services21. On the day of data collection, research assistants approached all women aged 18 years or older who were at the health facility seeking MNCH services (ANC, postnatal, well-, or sick-child care), screened those who were willing to participate and consented those who met study eligibility criteria. Those who were pregnant and/or had a child under 2 years old and consented to participate were asked to carry a client-flow form which was completed by service providers at various service delivery points during the visit. Women in active labor were excluded since we could not expect them to keep track of providers caring for them and the form during childbirth. The client-flow form allowed up to five healthcare workers to check off services provided on a standard list of possible services and note any referral recommendations. The client-flow forms were collected at the point where each client exited the health facility.\n\nSemi-structured, key informant interviews were conducted with local health authorities (district or sub-county officials), facility management and service providers to understand perceived benefits of PPFP integration, challenges in implementing integrated services, and the institutionalization of integrated services in the health system. In India, experienced qualitative interviewers from the International Institute of Health Management Research (IIHMR) conducted interviews. In Kenya, two independent consultants were hired and trained to conduct interviews. Key informants were selected on the basis of their involvement in supervising MNCH and PPFP services in the selected facilities. Each service provider was interviewed only once either as a facility health in-charge or about the PPFP services they provided. Providers were asked which service delivery unit they worked in (ANC, labor and delivery, postnatal care and/or child health). They were also asked to pick one of those units which they spend more time in and to answer subsequent questions about their work in that unit. At each site, qualitative researchers conducted interviews in a single day using structured interview guides designed specifically for each type of respondent: key informant, in-charge, or provider. Key informants and in-charges had to have a role in overseeing MNCH services in the facility including integration of PPFP. Service provider interviews were conducted in either Hindi or English in India, and solely in English in Kenya. Interviews lasted between 30 and 60 minutes and were conducted in private. While the study team discussed the concept of saturation with data collectors, they completed the number of interviews assigned, ensuring no overlap between quantitative survey and qualitative interview respondents. All interviews were recorded, transcribed, and, if necessary, translated to English prior to analysis.\n\nA total of 215 MNCH clients and 39 health care providers were surveyed, and 10 key informants and 33 facility informants interviewed, across the four study sites (Table 1).\n\nMedian age of participants in both countries was 23 years, range 18-42 years.\n\n* Interviews excluded from analysis after facilities sorted by level of integration (see Table 2)\n\nQuantitative data analysis. Research assistants entered quantitative data from client and provider surveys into REDCap, version v6.0.1., a web-based software for data capture and management. Descriptive analysis was conducted using SPSS Statistics 22 and Stata (version 12). The investigators carried out frequency distributions and cross-tabulations and reported the outputs in percentages. No statistical testing was done.\n\nFP integration and qualitative data analysis. We analyzed qualitative data using a grounded theory approach. Interview scripts were read iteratively and open/conceptual and open codes generated. We conducted the FP integration analysis in three phases. The first phase comprised of four steps, as follows: Step 1: quantitative data from client surveys, health worker surveys, and client-flow analysis by facility were collated using Microsoft Excel. We excluded two facilities from Kenya where we did not collect client flow data because they had fewer than five client respondents per service area. Step 2: three research team members separately analyzed the collated client and provider survey data to generate independent assessments of the level of PPFP integration within maternal and child health services, to develop an approach similar to previous studies22,23. To address disagreements between the independent assessment of level of integration, we further disaggregated data by service area within each facility (ANC, Labor and Delivery, PNC, Child Health) and jointly analyzed the level of PPFP integration within each service area of each facility, and then for the facility overall. In only two facilities were women having given birth in that visit interviewed. For each facility, analysis included the number of clients interviewed at the unit they initially sought care from (ANC, PNC or child health), and the percentage that indicated PPFP was discussed. Step 3: the number of providers surveyed, and their responses to the question as to how many among the last 10 clients seen in their ‘primary’ unit to whom they provided PPFP counseling or services (range 0–10). Step 4: client flow data were considered, specifically the number of clients who indicated that the purpose of the visit was ANC, labor and delivery, PNC or immunization/child health, as well as the number where the provider ticked having offered FP counseling or provision of a method.\n\nSimilar to previous studies22,23, we used an ordinal scoring system to characterize the level of service integration, where 1 = low levels of integration (0-29% of MNCH client visits included PPFP and survey data suggested little integration), 2 = moderate levels of integration (30–59% of visits included PPFP and survey data suggested some integration), and 3 = high levels of integration (60–100% of visits included PPFP and survey data suggested integration). In cases where there was not concordance between client flow and interview data, we relied on the client flow data as a more robust source. Independent rankings were then reviewed by all three researchers. There was insufficient data to consistently analyze level of PPFP integration in PNC and child health services. We thus used the levels of integration in ANC to disaggregate health facilities with high and moderate levels of integration and grouped these as well integrated (shown in dark gray in Table 2) and those with low-levels of integration we termed poorly integrated (shown in light gray in Table 2). The decision to group the middle tier of integration as “well integrated” was made on the assumption that this integration was unlikely to be by chance, but as a result of the program having some effect.\n\nKO03 and KO06 excluded because insufficient data for all units. Text indicates where on broad range of level of integration (30–100%). If there are two notations, it indicates differences in client flow and client survey data, where at least 5 clients were surveyed about that service area. Bold text indicates well-integrated care, non-bold indicates poorly integrated care.\n\n*Unlike the other service areas, integration of FP in antenatal care involves only FP information, counseling, possibly condoms and referrals. In other service areas, there should also be provision of a contraceptive method or intra-facility referral for provision.\n\nThe second phase of analysis involved reviewing the semi-structured interview transcripts to identify factors enabling and limiting integration of PPFP services at each facility and coded the text using a combination of thematic and free-coding in ATLAS.ti 7.5 (Scientific Software Development GmbH, Berlin, Germany). We reviewed qualitative findings across sites to identify characteristics common to well-integrated facilities separately from those that were poorly integrated.\n\nIn the third phase, we analyzed antenatal care client reports of privacy, interactions with staff, and waiting times to compare experiences at well-integrated and poorly integrated facilities by calculating percentages per site. We did not use the same approach for postnatal care or child health clients because of the small numbers of these respondents. To synthesize findings, we triangulated analyses described above, and examined trends in client, provider and management experiences at well-integrated and poorly integrated facilities in each study county, country, and overall.\n\nThe study was approved by the Johns Hopkins School of Public Health institution review board (No 5517), IIHMR’s Institutional Review Board in India, and the Kenya Medical Research Institute (KEMRI) Ethical Review Committee in Kenya. All participants provided oral informed consent, in accordance with approved research protocols, in order to avoid collecting personal identifiers for study participants.\n\n\nResults\n\nThis study assessed the extent to which PPFP is integrated in MNCH services and identified factors affecting service delivery in four distinct settings, two in Kenya and two in India. Kenyan sites included 2 hospitals (1 in Embu, 1 in Bondo) and 7 health centers (2 in Embu, 5 in Bondo), while all six facilities selected in India were hospitals. All facilities had been providing PPFP services for at least two years. Clients’ mean (SD) age was 24.4 (4.8) years, and the majority were married (100% in India; 80% in Bondo, and 85% in Embu). Clients’ level of education varied greatly; 60% of clients in India had completed secondary school or higher, compared to 80% of clients in Embu, Kenya and 20% of clients in Bondo, Kenya. Data from provider and client exit interviews are available on Open Science Framework20.\n\nTable 2 shows the level of PPFP integration across groupings of MNCH services at each facility: 1) within maternal health services (ANC or labor/delivery) and 2) within child health services (postnatal and well- or sick- child care services). Of the 13 facilities examined, only two (one from Kenya and one from India) were deemed to have a high level of integration in both groupings of MNCH services. There were six facilities (three from Kenya and three from India) that demonstrated a high level of service integration in either maternal health or child health services.\n\nBecause some determinants of integration may be a function of contextual factors, it is reasonable to expect facilities within the same national or subnational health system to be more similar to each other than to facilities in other areas. Our analysis revealed some similarities in levels of integration by facility type and service area, but scores were not consistent enough to draw strong conclusions about overall levels of integration achieved in all study sites. For example, in Jharkhand, India, PPFP appeared to be well integrated in maternal health services, but only one of three facilities had integrated PPFP in child health services. Health centers in Bondo, Kenya, integrated PPFP in child health services relatively well and performed better than the larger health facility that supports them (the sub-county hospital). Health facilities (a hospital and two health centers) in Embu, Kenya, showed the lowest degree of PPFP integration, while facilities in Bihar, India, showed the most variation between well- and poorly integrated services. Analysis was also limited by imbalance in the number of clients surveyed at each facility. It is worth noting that the number of clients surveyed at well-integrated facilities was at least four times greater than at poorly integrated facilities.\n\nAlthough levels of integration varied by service area and health facility, common themes were expressed in semi-structured interviews at both well-integrated and poorly integrated facilities. The themes were related to the importance of: (1) dedicating appropriate space for PPFP services, (2) considering health workforce composition, capacity, and motivation, and (3) ensuring consistent and affordable supply of a range of contraceptive methods. The influence of these factors on the level of PPFP integration were also evident in findings from provider and client surveys.\n\nAppropriate space for PPFP services. Space provisions, including appropriate spaces for PPIUD insertion, were frequently mentioned as factors enabling PPFP integration. Highly integrated facilities were more likely to have dedicated spaces for PPFP counseling, which may or may not be separate rooms but provide privacy and space for client and counselor. However, our findings also show that a dedicated space for PPFP services alone is not sufficient for successful integration of services, and that not all dedicated spaces adequately meet provider needs:\n\n“…the original plan did not have the integration aspect in mind. So the current existing room could be very small, like the one we are seated in... Now that [PPFP] is coming in, you may need an extra tray table for demonstration. You need a couch for examination and, if that space is not there, then you find it can be a challenge until the staff can generally say there is nothing we can do.” [Provider, Bondo]\n\nIn addition to not having dedicated spaces for PPFP services, poorly integrated facilities were characterized by chaotic environments:\n\n“It is in different room therefore they face problem in inserting IUCD…[T]here is no special room [for inserting PPIUD]. And most importantly, I want our counseling room to be improved. I mean when a client visits my room after meeting the doctor, then she must feel at ease here and can talk to us in relaxed state of mind...And here, also, you hear continuous noise to the point where, if someone tries to speak, nobody can listen clearly. This place is just besides the outpatient department; therefore, there is a continuous rush over here.” [Counselor, Bihar]\n\nAdditional infrastructure issues, such as the physical layout of a facility, may pose challenges to effective integration of PPFP services. Data collection team members shared that one Bihar facility layout required clients to navigate complicated pathways to various units; this facility was independently ranked as being poorly integrated.\n\nAll MNCH clients surveyed were asked about privacy concerns regardless of whether PPFP was discussed during their visit. A greater proportion of clients in Kenya than in India, reported acceptable levels of privacy (88.2% in Kenya versus 39.5% in India) and feeling comfortable discussing private/sensitive health concerns (84.7% in Kenya versus 45% in India). When comparing well-integrated and poorly integrated facilities in Kenya, the vast majority of clients at facilities in both categories reported that visual and auditory privacy were not a problem (92.7% at well-integrated facilities versus 87.3% at poorly integrated facilities; p=0.999). In India, by contrast, stark differences emerged between well- and poorly integrated facilities. At well-integrated facilities, the majority (69.7%) of clients reported privacy as a problem, compared with 20.0% at poorly integrated facilities (p =0.001), as shown in Table 3. In all sites, having concerns about visual/auditory privacy was correlated with concerns about discussing private/sensitive health concerns.\n\na In total, 215 clients were interviewed as indicated in Table 1 and responded to a series of “statement” questions: minor problem; major problem, or no problem. These responses were cross-tabulated with the variable on the level of integration at ANC (poor vs. well). Based on earlier analysis and categorization of the integration, some health facilities ended up NOT having a level of ANC integration being assigned to them, due to “inadequacy of available information”. Correspondingly, the responses of clients in these respective health facilities were “dropped” during cross-tabulation. In Kenya, 26 records were dropped, while in India, 23 records were dropped, for a total of 49 records.\n\nHealth workforce composition, capacity, and motivation. Having a dedicated, well-trained PPFP counselor on staff was another factor identified as contributing to successful integration of services, as it increases the likelihood that clients will have their PPFP needs addressed in the same visit. As an in-charge of a Jharkhand facility explained, “We have the counselor here specifically for counseling, and she is able to give enough time.” Well-integrated facilities were also more likely to have motivated staff willing and able to take on additional responsibilities. One Bihar provider noted, “I have learned by hearing others. And I made my mind that [counseling] is my responsibility, and I have to do it.”\n\nStaffing and wait time are often linked in provider statements, but providers who are motivated to implement integrated services may portray the issue differently. For example, a service provider from a well-integrated facility noted:\n\n“[Integration] is good because everybody can provide the services, and the client does not need to wait for one person or go to a particular service provision place for the [family planning], and that has really helped in reducing the [loss] of clients, as well as reducing the time they take when they come for those services.” [Provider, Bondo]\n\nPoorly integrated facilities were often staffed by individuals either unable or unwilling to take on responsibilities beyond what they saw as their traditional job tasks. In many cases, the facilities ascribed this to shortages of health workers, especially in rural and remote areas. A nurse in Bihar, India, explained that at the facility where she works, “No one fulfills his/her responsibilities appropriately due to scarcity of staff.” Additionally, service providers in Embu echoed this constraint, explaining that the integration of PPFP counseling has increased wait times for clients, generating complaints that negatively impact job satisfaction. As one Bondo In-charge explained, “If the staffing improves, then I think the services will also improve more because sometimes someone may not have all time…like now you see [a family planning patient] is waiting for you…so sometimes one may be tempted to move faster than it should be and that may compromise the service.”\n\nSome providers felt that long client wait time was a disadvantage of integrating PPFP counseling with MNCH services. This concern was not supported by ANC clients in Kenya, where only 35.7% of clients in well-integrated facilities and 38.0% in poorly integrated facilities reported wait time as a problem. However, in India, the majority of ANC clients (66.7%) in highly integrated facilities reported wait time as a problem as compared with only 26.7% of ANC clients at poorly integrated facilities, as shown in Table 3. We note that all well-integrated facilities in Kenya were health centers, whereas all sampled India facilities were hospitals.\n\nA related but distinct issue around staffing relates to provider training and preparation to offer integrated services. As a facility manager in Embu, Kenya, explained, “What we needed most was training of which we still need up to now. As you have heard, we had one person trained on PPIUD who now left, so we would like more people to be trained.” Providers also saw training on PPFP counseling and service provision as a professional development opportunity and noted the integration of counseling in other service areas provided clients with opportunities for closer interaction with service providers, resulting in increased trust in the health system:\n\n“So things are going well here in district hospital. However, one portion, in particular, is weak from our side and [that] is counseling. Counseling, which is related to ANC period. Counseling is weaker; therefore, we are not able to effectively facilitate the decision-making of mothers visiting our facilities. Mothers who visit the facilities for delivery needed time for mind-making, for taking decisions. So if our counseling is improved, then our overall performance would be improved.” [Key Informant, Jharkhand]\n\nAlthough we only surveyed 17 ANC providers, responses found that higher proportions of providers in well-integrated facilities reported having knowledge and skills to provide both PPFP counseling and services, compared to providers in poorly integrated facilities.\n\nConsistent, affordable supply of contraceptive methods. Key informants and facility staff in Kenya and India highlighted the importance of access to and affordability of contraceptive commodities. Another Bihar facility in-charge also mentioned the role of educational materials: “We were not able to tell about the methods, and, also, we didn’t have this kit so we were not sure whether [the mother] understood about the methods or not.” Because the type of family planning supplies needed vary across service areas, it was challenging to assess differences in commodity availability within our relatively small sample of well- and poorly integrated facilities.\n\nAt some facilities, staff reported that increased demand for contraceptives generated by integrating services initially resulted in stock-outs, but, over time, they were able to establish systems to address this issue. Others reported occasional stock-outs or little information on availability of commodities. Facilities with mid to high levels of PPFP integration tended to have a well-functioning supply chain management system while some poorly integrated facilities did not monitor their supply levels. As a key informant in a poorly integrated facility in Bihar explained, “Yes, there have been [stock-outs] sometimes. I cannot tell you much about this, frankly speaking as many times our staff could not check these things.”\n\nIn some facilities, commodities may be available, but user fees remained a barrier to contraceptive uptake. For example, a health worker in Embu, Kenya noted “Some women are not able to afford [PPFP], especially the long-term methods.” Similarly, a provider in Bihar, India explained that despite efforts to cover costs, the health system has not been able to fully eliminate user fees: “Is it [commodity costs] covered 100%? Not exactly, we covered 50% now and trying to cover 100% in near future.” Furthermore, a provider in Bihar also reported client concerns over the cost of IUD removals, which she sought to allay during counseling. The issue of costs for clients seemed to appear only in poorly integrated facilities in both Kenya and India.\n\nContext matters: Additional factors affecting the success of integration efforts. In addition to the issues discussed above, two context-specific themes were raised throughout semi-structured interviews in Jharkhand and Bihar, India, as major factors affecting the success of integration efforts: provider-client power dynamics and demand-side financing programs.\n\nIn both Bihar and Jharkhand, providers articulated that they think some challenges to PPFP uptake are due to what they perceive as clients’ religious beliefs and education levels. In Jharkhand, a head nurse described the clients as “backwards,” “uncivilized,” and “uneducated.” In Bihar, providers counsel religious clients that “not spacing and caring for kids is a sin against God.” Providers in both Jharkhand and Bihar opined that Muslim women refuse PPFP because of their cultural beliefs, specifically citing that if a woman is operated on, she will not be placed for burial after death. Additionally, providers highlighted that how they counsel a woman is often based on the number of children the woman already has rather than the woman's expressed preferences or needs. As one counselor in Bihar explained, “We try to motivate those for sterilization who have two children.” While these attitudes are independent from integration efforts, integration implies an increase in number of providers discussing family planning with clients and that these providers need additional guidance on the rights of family planning clients to make voluntary and autonomous decisions about contraception.\n\nIn addition, many key informants and facility staff in both states of India mentioned the effects of financial incentives, specifically the JSY conditional cash transfer program, on PPFP uptake. Incentives are frequently used in India to assist in motivating clients as well as health workers. The role of incentives varied across Bihar and Jharkhand, but respondents in all sites agreed that they are very important. In Jharkhand, an auxiliary nurse-midwife highlights how the incentives have shifted community norms and helped motivate other clients to come: “we keep on telling one thing, so people understand and after that they tell others also. Then they are also getting money [JSY], so people come.” However, whether these incentives are in the best interest of the client is cloudy.\n\nSemi-structured interviews conducted in Kenya did not yield as rich descriptions of factors affecting the success of service integration efforts as interviews conducted in India. However, one provider in Bondo expressed a sense of teamwork with the sub-county health management team:\n\n“So all the commodities, the infrastructure, the equipment, the furniture and, of course, that consistent support supervision. Even as we make report, we forward [the report to] them and they are able to share with us. This…is the support in itself because, maybe during that preparation of that report, there is a glaring error that [is noticed] and I missed it. So the sub-county record person during that editing time, just a phone call, they have done this [asked about data, and corrected an error]. [Provider, Bondo]\n\nThis teamwork approach was also expressed by providers resolving commodity issues or coordinating with mobile teams to offer female sterilization at their facility.\n\n\nDiscussion\n\nDespite very different contexts, the factors identified as contributors or barriers to successful integration of PPFP in MNCH services were remarkably similar in both India and Kenya. Common themes identified across settings related to (1) dedicating appropriate space for PPFP services, (2) considering health workforce composition, capacity, and motivation, and (3) ensuring consistent and affordable supply of a range of contraceptive methods.\n\nThese findings are consistent with emerging evidence from other settings. Integration of health services is often assumed to be an efficient strategy for increasing access to care and a “diagonal approach to building primary healthcare” has been espoused24. Technical experts generally agree that optimal delivery of PPFP requires: taking advantage of existing contact points; direct provision of PPFP counseling or services during maternal, newborn, and child health (MNCH) consultations; or facilitated referrals to a co-located family planning provider6,25. Without interventions to coordinate the care provided, however, co-locating services in a facility has not been shown to increase the use of multiple services26. For example, in Uttar Pradesh, India, only a minority of antenatal and postnatal care clients receive advice about PPFP or birth spacing; as a result, unmet need for family planning has not decreased27,28. Further, a systematic review of integrated HIV and family planning services noted that facilitated referrals between co-located services were difficult to implement without a solid health system foundation, and weak fidelity to the designed service integration intervention may have led to lower than expected family planning uptake29. Changing the organization of services for the purposes of integration requires one or more interventions, such as training, new tools, supervision, and performance improvement. At the same time, fully integrating PPFP services across the continuum of care may have trade-offs for resource allocation and attention to other aspects of service quality8.\n\nRespondents from well-integrated Kenyan facilities cited provider competence and availability of FP commodities as facilitators, whereas poorly integrated facility respondents cited inadequate space, staff shortages, workload, end-user costs, and commodity stock-outs as barriers. While commodities are necessary for any service, integrated or not, shortages act as a disincentive to integrate services as that may exacerbate the problem of stockouts. Respondents from well-integrated facilities frequently complained about lack of space, yet they seemed to work around this challenge, indicating factors beyond space are critical for successful integration. Providers from well-integrated Indian facilities described having a room for the dedicated counselor, motivation to provide client-centered care, providers who believe in PPFP benefits, acceptance of workload, training for staff, continuous supply of FP commodities, and incentives for PPIUD insertions as favorable. Service providers in poorly integrated facilities in India said not all client costs were covered and reported low motivation (e.g., PPFP counseling was not their job), although underlying reasons for variances in provider motivation within the same system could not be ascertained in this study. Other researchers have noted that integration of physical and human resources (‘structural integration’) is insufficient to ensure that clients consistently receive integrated services (‘functional integration’)30.\n\nWithin geographic locality, program support was generally similar, yet the level of integration of PPFP within maternal health services or child health services varied. Of note, one Jharkhand facility has gone beyond initial program interventions to integrate PPFP strongly into child health services. Facilities in Bihar had the most variation in level of integration. In that state, qualitative results suggested that providers were much more motivated by incentives.\n\nThis findings of this study are in line with the findings of Mayhew et al., that there is considerable heterogeneity in levels of integration across their study sites in Africa, including Kenya30. The emphasis on incentives in Bihar could also imply that counseling is more systematic for women in labor and delivery and the immediate postpartum period, with potential missed opportunities in ANC. We cannot reliably report on the level of service integration or how consistently PPFP is integrated in a childbirth visit from our data. Nevertheless, quality PPFP should begin with ANC as women often need time to consult and consider their contraceptive method of choice31. The opportunity to do so was more evident in our sample of facilities in Jharkhand. The ethical issues associated with incentives have been noted in the literature32.\n\nIn Embu, Kenya, the level of service integration was unsurprisingly lower than in other sites, most likely due to the short duration of project support and the elapsed time between end of project and this assessment—about 5 years. Furthermore, attrition of trained providers affected service integration in those sites, particularly the smaller facilities. Sweeney and colleagues, in their study of HIV, sexual and reproductive health and postnatal care integration, reported on the slow pace of change to integrate health services33. This implies that a short intervention period to effect change in service integration may not bring about lasting change as was seen in Embu. In comparison, integration was stronger in Bondo health centers, which have fewer staff who tend to provide a greater number of services, as compared to hospitals which have more staff who tend to work in distinct units providing a smaller number of services. Integration of services was also stronger in postnatal or child health services, as compared to ANC or labor and delivery services, which may reflect the ease of service integration when the program emphasizes integrating PPFP into infant and young child services.\n\nMost of the poorly integrated facilities had staff who reported high workloads, lack of skills to insert PPIUDs, frequent stock-outs of family planning commodities, and client privacy challenges. On the other hand, providers in well-integrated facilities in India and Kenya reported greater commitment to high-quality services for clients. This contrasts with the study by Church et al., which found that providers rarely saw personal benefits of integrated care HIV and reproductive health care, perceiving benefits mostly for clients34. Clients seeking ANC services in well-integrated facilities in Kenya did not report concerns with wait time or privacy. In well-integrated facilities in India, however, these factors, along with lack of concern for client autonomy and method choices, were reported as minor or major problems. Other studies in India have found that providers negatively judge clients’ education, family planning needs, and ability to understand contraceptive options, thereby imposing unnecessary barriers to acquiring family planning methods35. Nevertheless, provider reports of barriers to integration in this study are not conclusive given that time-motion studies show that health workers exaggerate time needed for a given health intervention30.\n\nWhen reviewed through the lens of poorly versus well-integrated services, analysis of client responses on different aspects of service quality (privacy, comfort, wait time, staff treatment of clients) provided interesting insights. For example, a higher proportion of clients in well-integrated facilities in India, compared to those in poorly integrated facilities, reported problems with various aspects of services, which may seem striking. However, clients from poorly integrated facilities were less likely to be exposed to PPFP services, which are often considered to be a sensitive issue in our Indian study sites. Similarly, clients from well-integrated facilities were more likely to receive PPFP services, which may have led to increased concerns of privacy and discomfort discussing sensitive issues. Greater frequency of concerns about a lack of privacy and comfort discussing PPFP, as reported by clients in India compared with Kenya, may be due to perceived cultural sensitivity about family planning overall, which have been reported elsewhere36.\n\nIn India, the finding that wait times were more likely to be perceived as a problem in well-integrated facilities is also not surprising given high client volumes in these facilities. This finding is also consistent with case studies of integration in sub-Saharan Africa37. Of concern in the Indian sites is the disproportionate number of clients reporting mistreatment by staff as a problem in well-integrated facilities, compared with poorly integrated facilities. Furthermore, the influence of monetary incentives may induce certain providers to apply more pressure on women to adopt a contraceptive method, which would amount to coercion and against the principle of informed choice when providing family planning services.\n\nAs an exploratory study attempting to identify trends in health facility and service delivery characteristics across four distinctly different program settings, our analysis is not without limitations. Our analysis of levels of integration relies heavily on an assessment done through a client-flow and data collection activity that was not feasible to conduct with women attending facilities for childbirth so may not accurately represent levels of PPFP integration across all MNCH services. In addition, the 1-week duration of observation may not accurately represent levels of integration over time. We acknowledge that systematic observation of provider-client interactions in a clinic setting over a longer time period would probably have generated more robust data. Second, the number of facilities sampled in each location was small and precluded generating insights about the type or size of facility that may be more amenable to integrating services. The purposive sampling also implies that our results may not be generalizable. Third, our interview data focused heavily on ANC clients as opposed to immediate postpartum clients, as the study team felt it burdensome to ask women to participate in an hour-long interview immediately after giving birth. However, a substantial number of PNC clients were interviewed at the child health services, thus perspectives of postpartum women were included in our analysis and the use of a mixed method approach with data collection tools designed to explore many aspects of service provision allowed us to gather valuable insights in settings where there is limited evidence on effective strategies for service integration. We also recommend additional research on establishing measures of service integration that should be validated in randomly selected facilities.\n\nOur findings highlight the complexities that must be considered in integration of health services. Policymakers contemplating increased integration should thus base their strategies on an understanding of the diversity in the health facilities they seek to affect. Policymakers also need new ways to measure whether integration of services is actually taking place as prescribed. Our findings showed that, even after program support to integrate services, actual levels of service integration varied greatly.\n\n\nConclusions\n\nThrough triangulation of data from clients, providers and a client flow tool, our study sought to innovatively measure and qualitatively understand the level of PPFP integration with antenatal and child health services and factors that enabled or limited this integration at facilities that receive program support to integrate PPFP into maternal and child services. The factors identified were common across Kenya and India. Common themes emerged from interviews with providers and facility in-charges at both well- and poorly integrated facilities: issues influencing integration included allocation of physical space for PPFP service delivery, health workforce composition and capacity, and commodity availability, having staff to manage the combined services, client wait times, the organization of services to ensure that commodities are readily available, and as well as duration and nature of related program support. Service providers in well-integrated facilities are more likely to express motivation for client-centered, integrated care. Our analysis contributes to the limited literature on the integration of PPFP with maternal and child health services. The results are not clear cut but show the complexity and nuances of effectively implementing and scaling up integrated services. Better measures are needed to verify whether services are integrated as prescribed by national policies.\n\n\nData availability\n\nDryad: Data from: Postpartum family planning integration with maternal, newborn, and child health services: a cross-sectional analysis of client flow patterns in India and Kenya. https://doi.org/10.5061/dryad.1131319. The base dataset for client flow data is available in SAV, DTA and CSV formats. A codebook is available in DOCX format. Data were generated in a previous study18.\n\nOpen Science Framework: PPFP Integration in India and Kenya. https://doi.org/10.17605/OSF.IO/5TFA720. The following underlying data files are available:\n\nProvider exit interviews (Tool 3b_FINAL.csv) with codebook (Tool 3b_CODEBOOK_SEED_2.csv).\n\nClient exit interviews (Tool 5a_FINAL.csv) with codebook (Tool 3b_CODEBOOK_SEED_2.csv).\n\nIn addition, we collected, transcribed and translated, where appropriate, in-depth interviews (qualitative data). The study team feels it would be impossible to completely de-identify qualitative interviews. Furthermore, since the protocol approved by 3 ethical review committees (United States, India and Kenya) did not allow for sharing of qualitative data, we are not able to provide this dataset. If researchers would like to use the qualitative data, to either replicate our analysis or for other purposes, they may contact the corresponding author. They will be asked to sign a data use agreement prior to receiving any transcripts. We may also request that they obtain permission from the corresponding in-country IRBs.\n\nOpen Science Framework: PPFP Integration in India and Kenya. https://doi.org/10.17605/OSF.IO/5TFA720. The following extended data files are available:\n\nKey Informant Interview Guide – Tool 1 (PPFP_Integration_IRB5517_Tool1-KI_IntGde_English_v3_2014May06.docx)\n\nFacility In-Charge Interview Guide (bilingual) – Tool 2 (PPFP_Integration_IRB5517_Tool2-Fac_InChg_IntGde_Bilingual_India_v2_2014May06.docx)\n\nFacility-Based Provider Interview Guide – Tool 3a (PPFP_Integration_IRB5517_Tool3a-Fac_Prov_IntGde_Bilingual_India_v2_2014May06.docx)\n\nIndia: Facility-Based Provider Survey – Tool 3b (PPFP_Integration_IRB5517_Tool3b-Fac_Prov_Survey_India_v2_2014May02.docx)\n\nKenya: Facility-Based Provider Survey – Tool 3b (PPFP_Integration_IRB5517_Tool3b-Fac_Prov_Survey_Kenya_v2_2014Jun05.docx)\n\nIndia: Client Survey – Tool 5a (PPFP_Integration_IRB5517_Tool5a-Client_Survey_India_v2_2014May21.docx)\n\nKenya, Luo: Client Survey – Tool 5a (PPFP_Integration_IRB5517_Tool5a-Client_Survey_Kenya_v2_2014Jun05_Luo_TRACKED.doc)\n\nKenya, Swahili: Client Survey – Tool 5a (PPFP_Integration_IRB5517_Tool5a-Client_Survey_Kenya_v2_2014Jun05_Swahili_TRACKED.docx)\n\nKenya, English: Client Survey – Tool 5a (PPFP_Integration_IRB5517_Tool5a-Client_Survey_Kenya_v2_2014Jun05_TRACKED.docx)\n\nIntegration Client Flow Form – Tool 5b (PPFP_Integration_IRB5517_Tool5b-Client_Flow_ENG_2014May03.pdf)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThis study was conducted and its publication made possible through support from the United States Agency for International Development, as part of the Maternal and Child Health Integrated Program (MCHIP) Award (Number GHS-A-00-08-00002-000) and subsequent Maternal and Child Survival Program Award (Number AID-OAA-A-14-00028). The views in this paper represent those of the authors alone and not those of the US government.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe study team acknowledges Jim Ricca and Judith Robb-McCord for contributions in the design and, in Ms Robb-McCord’s case, support to field work of this study. In India, data collection activities were conducted by the International Institute of Health Management Research, under the leadership of B.S. Singh, with support and oversight from MCHIP study team members. Additional data collectors and field supervisors supported work in Kenya, including Angella Ongutu, Joygrace Muthoni and Charles Waka, who also served as REDCap data manager.\n\n\nReferences\n\nStover J, Ross J: Changes in the distribution of high-risk births associated with changes in contraceptive prevalence. BMC Public Health. 2013; 13 Suppl 3: S4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKozuki N, Lee AC, Silveira MF, et al.: The associations of birth intervals with small-for-gestational-age, preterm, and neonatal and infant mortality: a meta-analysis. BMC Public Health. 2013; 13 Suppl 3: S3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCleland J, Conde-Agudelo A, Peterson H, et al.: Contraception and health. Lancet. 2012; 380(9837): 149–56. PubMed Abstract \| Publisher Full Text\n\nMoore Z, Pfitzer A, Gubin R, et al.: Missed opportunities for family planning: an analysis of pregnancy risk and contraceptive method use among postpartum women in 21 low- and middle-income countries. Contraception. 2015; 92(1): 31–9. PubMed Abstract \| Publisher Full Text\n\nGaffield ME, Egan S, Temmerman M: It's about time: WHO and partners release programming strategies for postpartum family planning. Glob Health Sci Pract. 2014; 2(1): 4–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization (WHO), United States Agency for International Development (USAID), Maternal and Child Health Integrated Program (MCHIP): Programming strategies for postpartum family planning. World Health Organization, 2013. Reference Source\n\nVernon R: Meeting the family planning needs of postpartum women. Stud Fam Plann. 2009; 40(3): 235–45. PubMed Abstract \| Publisher Full Text\n\nDudley L, Garner P: Strategies for integrating primary health services in low- and middle-income countries at the point of delivery. Cochrane Database Syst Rev. 2011; (7): Cd003318. PubMed Abstract \| Publisher Full Text\n\nAli M, Seuc A, Rahimi A, et al.: A global research agenda for family planning: results of an exercise for setting research priorities. Bull World Health Organ. 2014; 92(2): 93–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuhlmann AS, Gavin L, Galavotti C: The integration of family planning with other health services: a literature review. Int Perspect Sex Reprod Health. 2010; 36(4): 189–96. PubMed Abstract \| Publisher Full Text\n\nSonalkar S, Mody S, Gaffield ME: Outreach and integration programs to promote family planning in the extended postpartum period. Int J Gynaecol Obstet. 2014; 124(3): 193–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCleland J, Shah IH, Daniele M: Interventions to Improve Postpartum Family Planning in Low- and Middle-Income Countries: Program Implications and Research Priorities. Stud Fam Plann. 2015; 46(4): 423–41. PubMed Abstract \| Publisher Full Text\n\nAtun R, de Jongh TE, Secci FV, et al.: Integration of priority population, health and nutrition interventions into health systems: systematic review. BMC Public Health. 2011; 11: 780. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShade SB, Kevany S, Onono M, et al.: Cost, cost-efficiency and cost-effectiveness of integrated family planning and HIV services. AIDS. 2013; 27 Suppl 1: S87–92. PubMed Abstract \| Publisher Full Text\n\nMutemwa R, Mayhew S, Colombini M, et al.: Experiences of health care providers with integrated HIV and reproductive health services in Kenya: a qualitative study. BMC Health Serv Res. 2013; 13: 18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCresswell JA, Plano Clark V: Designing and Conducting Mixed Methods research. Thousand Oaks, CA: Sage Publications; 2007. Reference Source\n\nCooper C, Ogutu A, Matiri E, et al.: Maximizing Opportunities: Family Planning and Maternal, Infant, and Young Child Nutrition Integration in Bondo Sub-County, Kenya. Matern Child Health J. 2017; 21(10): 1880–1889. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMackenzie D, Pfitzer A, Maly C, et al.: Postpartum family planning integration with maternal, newborn and child health services: a cross-sectional analysis of client flow patterns in India and Kenya. BMJ Open. 2018; 8(4): e018580. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMackenzie D, Pfitzer A, Maly C, et al.: Data from: Postpartum family planning integration with maternal, newborn, and child health services: a cross-sectional analysis of client flow patterns in India and Kenya. Dryad Digital Repository. 2018. http://www.doi.org/10.5061/dryad.11313\n\nPfitzer A, Kabue M: PPFP Integration in India and Kenya. 2019. http://www.doi.org/10.17605/OSF.IO/5TFA7\n\nBirdthistle IJ, Mayhew SH, Kikuvi J, et al.: Integration of HIV and maternal healthcare in a high HIV-prevalence setting: analysis of client flow data over time in Swaziland. BMJ Open. 2014; 4(3): e003715. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCoker R, Balen J, Mounier-Jack S, et al.: A conceptual and analytical approach to comparative analysis of country case studies: HIV and TB control programmes and health systems integration. Health Policy Plan. 2010; 25 Suppl 1: i21–31. PubMed Abstract \| Publisher Full Text\n\nTopp SM, Chipukuma JM, Chiko MM, et al.: Integrating HIV treatment with primary care outpatient services: opportunities and challenges from a scaled-up model in Zambia. Health Policy Plan. 2013; 28(4): 347–57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGounder CR, Chaisson RE: A diagonal approach to building primary healthcare systems in resource-limited settings: women-centred integration of HIV/AIDS, tuberculosis, malaria, MCH and NCD initiatives. Trop Med Int Health. 2012; 17(12): 1426–1431. PubMed Abstract \| Publisher Full Text\n\nPfitzer A, Mackenzie D, Blanchard H, et al.: A facility birth can be the time to start family planning: postpartum intrauterine device experiences from six countries. Int J Gynaecol Obstet. 2015; 130 Suppl 2: S54–61. PubMed Abstract \| Publisher Full Text\n\nLawn S, Lloyd A, King A, et al.: Integration of primary health services: being put together does not mean they will work together. BMC Res Notes. 2014; 7: 66. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAchyut P, Mishra A, Montana L, et al.: Integration of family planning with maternal health services: an opportunity to increase postpartum modern contraceptive use in urban Uttar Pradesh, India. J Fam Plann Reprod Health Care. 2016; 42(2): 107–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYadav D, Dhillon P: Assessing the impact of family planning advice on unmet need and contraceptive use among currently married women in Uttar Pradesh, India. PLoS One. 2015; 10(3): e0118584. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilcher R, Hoke T, Adamchak SE, et al.: Integration of family planning into HIV services: a synthesis of recent evidence. AIDS. 2013; 27 Suppl 1: S65–75. PubMed Abstract \| Publisher Full Text\n\nMayhew SH, Ploubidis GB, Sloggett A, et al.: Innovation in Evaluating the Impact of Integrated Service-Delivery: The Integra Indexes of HIV and Reproductive Health Integration. PLoS One. 2016; 11(1): e0146694. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar S, Sethi R, Balasubramaniam S, et al.: Women's experience with postpartum intrauterine contraceptive device use in India. Reprod Health. 2014; 11: 32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBellows BW, Conlon CM, Higgs ES, et al.: A taxonomy and results from a comprehensive review of 28 maternal health voucher programmes. J Health Popul Nutr. 2013; 31(4 Suppl 2): 106–28. PubMed Abstract\n\nSweeney S, Obure CD, Terris-Prestholt F, et al.: The impact of HIV/SRH service integration on workload: analysis from the Integra Initiative in two African settings. Hum Resour Health. 2014; 12: 42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChurch K, Wringe A, Fakudze P, et al.: The relationship between service integration and client satisfaction: a mixed methods case study within HIV services in a high prevalence setting in Africa. AIDS Patient Care STDS. 2012; 26(11): 662–73. PubMed Abstract \| Publisher Full Text\n\nCalhoun LM, Speizer IS, Rimal R, et al.: Provider imposed restrictions to clients’ access to family planning in urban Uttar Pradesh, India: a mixed methods study. BMC Health Serv Res. 2013; 13(1): 532. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRyman TK, Wallace A, Mihigo R, et al.: Community and health worker perceptions and preferences regarding integration of other health services with routine vaccinations: four case studies. J Infect Dis. 2012; 205 Suppl 1: S49–55. PubMed Abstract \| Publisher Full Text\n\nCairns KL, Perry RT, Ryman TK, et al.: Should outbreak response immunization be recommended for measles outbreaks in middle- and low-income countries? An update. J Infect Dis. 2011; 204 Suppl 1: S35–46. PubMed Abstract \| Publisher Full Text" }	[ { "id": "46707", "date": "10 Jun 2019", "name": "Priya Nanda", "expertise": [ "Reviewer Expertise Health Systems research and gender issues." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very interesting study that draws its interpretations from a very small sample in Kenya and India. They compare the state on integration and quality of services across different size facilities in each study sites. The data is biased as it draws on high client load in well integrated facilities than poorly integrated facilities. Providers who were available were interviewed but there is no information whether these are the providers who were also trained in PPFP.\n\nIt is not clear why the research team did not use facility client flow data over the last 3-4 weeks as a robust way to understand flows rather than through observation during the says of study visit.\n\nI like the approach of hierarchy of evidence and when there is disagreement between expert analyse and client flow giving preference to client flow data. Can the researchers explain when and in what instances did these disagreements happen? What might be other factors that the experts were looking at—this might help in understanding what are other metrics for well integration that the study could have looked at?\nThe definition of integration in this study may be limited. Would the authors like to reflect on how else they may have defined and measured it? For example they look at whether the client at ANC or other departments is getting FP counselling. What about capacity of counsellor to counsel on PPFP—is that a given that its is good in well integrated facilities? Is there more informed choice and method availability in well integrated facilities?\nIt would be helpful if the authors provide briefs on how scoring was done for integration. I see that they looked at wait times and privacy but its not clear to me whether these were attributes of well integrated or outcomes at well integrated facilities?\nWhen a facility is badly integrated, does it get lower client load—is there a way to say that from the experience of well and medium integrated facilities? Is it possible to get high client load at facilities that are more crowded but yet are badly integrated where client flow pathways are more complicated?\nIn this study, its difficult to discern what went into the analysis as a definition of integration and what from the analysis is coming out as a determinant of integration?\n\nThe results are interesting yet the methodology is unclear and seems a bit bias-what the authors report as determinants are also definitions of what is coded as well integrated? Bihar in fact shows contrary results where privacy and waiting times are worse in well integrated facilities. These results needs their own discussions—its possible that well integrated services take longer time but privacy cannot be lower in well integrated facilities? Or it can if integration is only about offering services at different points of contact.\nIn order to contextualize the information from respondents, it would be useful if the qualitative information is cited from coming from a well or poorly integrated facilities. For example, in facilities where providers are talking about poor counselling or poor training, are those also facilities that were coded as poorly integrated?\n\nThe article brings out issues of provider bias in the section on context. This is a key issue that needs to emphasised, as they clearly suggest that integrated services do not necessarily address provider bias or rights adequately.\n\nThe authors discuss the limitations of the study well which is one reason I am not bringing it up in my review. However their discussion draws heavily from other studies, as this study itself presents inconclusive and mixed evidence. I think the study would be stronger if we started the analysis with fixed parameters on which questions were asked and then triangulated the qualitative data to match whether the fixed parameters for well integrated match with qualitative analysis of attributes of good quality services (which may or may not have high correlation with integration).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5145", "date": "13 Jan 2020", "name": "Anne Pfitzer", "role": "Author Response", "response": "Thank you for your thoughtful review. In response to the issue of bias because higher client load in well-integrated facilities than poorly integrated ones: We should clarify that we had more clients surveyed in Indian well-integrated facilities, but this is not necessarily indicative of client loads. However, this is an interesting observation nevertheless. The purposive selection of facilities clearly had limitations, but client load was not a criterion for the study (although high volume of deliveries was a criteria for PPFP program support). While this may introduce bias, we were surprised about this finding as we would think that facilities with higher loads would have more difficulty with integration. We did find we had to make a correction and clarify that the issue of client respondents was specific to India, not Kenya. Regarding information as to whether provider interviewed were trained in PPFP: we asked providers whether they had confidence in their abilities, whether they had the skills to do it, and whether they received sufficient support, but not if they were trained. We chose to report on more direct indications of provider performance and did not examine relationship between self-reported training experience, confidence, skills, support and level of integration. For the client flow methods, we chose one week as we felt it would address any fluctuations due to weekly occurrences, such as market days. While more client flow data might have been more robust, this was the amount of time feasible within project resources. On the approach of hierarchy of evidence and analysis of levels of integration: additional text and text box have been added to provide further detail and transparency in classification of facilities. Disagreements occurred in cases where provider and client reports were inconsistent with each other and small numbers of respondents presented challenges for comparison; this was addressed by establishment of decision rules regarding inclusion criteria and hierarchy of data sources. The reviewer makes a valid comment about the limitations of looking strictly at integration of services without also addressing the quality of services, which would have required an observation. We do not have such information, only that all facilities received roughly equivalent program support within the programs described in the paper. On the issue of wait times and privacy, we should also note that wait times and privacy were not part of the determination of integration, but we sought instead to understand whether the level of integration had an effect on clients’ reporting of these attributes of services. But in reference to a later comment, we have added a bit more interpretation of the findings regarding privacy issues in the discussion section. Indeed our findings suggest that integration and quality are not entirely correlated, but this may be partly because a comparison of the effects of integration on quality is an imperfect one as we didn’t ask clients a counterfactual of whether they would have preferred longer wait times for example in exchange for additional services provided during the visit. The reviewer’s questions about levels of integration and client load are interesting questions. However, we do not feel our dataset can answer them.We appreciate the need to contextualize the qualitative responses, so we went back to the transcripts, in order to re-identify all the quotes and classified them. We have thus clarified whether they were from well-integrated or poorly integrated facilities. In the process, we had to make additional corrections when quotes were mislabeled. In one or two cases, we made cuts because some quotes may have been misinterpreted when segmented out of context. The review also makes a good point about provider bias related to context. We have added a sentence about bias as a separate issue to integration. We found the feedback about the questions asked very helpful and have added a text box outlining the questions used in the analysis of integration. As noted, we clearly outline the limitations of this study and highlight complexities to be considered in future evaluations of family planning integration in other health services." } ] }, { "id": "51879", "date": "27 Aug 2019", "name": "Marina A.S. Daniele", "expertise": [ "Reviewer Expertise Reproductive health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study is a timely and important effort to expand on the evidence base for successful PPFP integration with maternal and newborn health services. The attempt to evaluate programmatic experiences in a systematic way is commendable.\nThe researchers have a good knowledge of existing evidence and gaps and display this clearly in the introduction and discussion.\nThe methods section begins with an appropriate context setting section, describing integration programmes in the study sites. As done here, it is often useful for triangulation purposes to combine qualitative and quantitative methods. The client flow method used appears to be robust and cost-effective. The semi-structured interviews also appear to be sound in design and quality. As pointed out by the authors themselves in the discussion, observation of care would have provided important insights.\nHowever, I have doubts concerning the quality of the data generated from the provider and client surveys. The numbers are fairly small, and there is a notable risk of courtesy bias. In this regard, I am particularly curious about the extent to which providers, especially, were aware that the researchers were assessing family planning integration, and whether they felt they could be completely honest about the extent to which they did or did not provide PPFP services. It would be useful for the authors to comment on these issues in the discussion. The area I am most uncertain about is the ability of the data on privacy, waiting time and interactions with staff to support the kind of analysis presented. The numbers appear to be very small, so I am not sure that they support quantitative comparisons between facilities. Qualitative interviews with clients might have been a better way to explore their experience of care.\nIn addition, it is difficult to express a full opinion about the soundness of all the analysis done because of a somewhat confusing presentation of methods and results. I had to read the methods section several times in order to understand what was done, with what purpose, and in what order. Sentences describing the purpose of each phase, and step, should precede those describing which type of data was considered in turn.\nThe development of an integration score for facilities is an essential part of the analysis, but different (more or less collated or aggregated by service area) classifications of integration levels are described in the methods, presented in Table 2, and referred to in the results section. I also could not find a clear explanation about why, out of 15 facilities in the sample, integration scores are only presented for 13.\nWhile containing interesting findings, the results section could be reorganised to be more clear, perhaps with more correspondence with the analysis phases as described in the methods, e.g. into three subject areas rather than two, the third being client experience. As it is, the second section of the results (Factors influencing success) appears quite long and difficult to navigate. However, I am not certain that the parts on client experience should be retained, given the reservations about data quality that I expressed above.\nAlthough, it could benefit from some streamlining and re-organisation, the Discussion reflects the strengths and weaknesses of the study and discusses the findings in the light of relevant contextual factors which aid the interpretation. The piece could be improved by giving more prominence to the paragraphs discussing what the implications for policymakers and future researchers are.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5118", "date": "13 Jan 2020", "name": "Anne Pfitzer", "role": "Author Response", "response": "Thank you for your acknowledgement of the value of reporting on program evaluations. We also appreciate the reviewer taking time to provide a summary of the paper. We added a sentence to the Discussion section, noting potential for courtesy bias in provider interviews. The consent form did describe the purpose of the study: “We are trying to understand how family planning is included with other health services at this facility particularly maternal, newborn child health and nutrition services.“ and …”ask you a series of questions related to maternal, newborn, child health and nutrition services offered at this facility, the inclusion of family planning in these services and what impact the inclusion of family planning has had ….a) on these services and in general at this facility (providers, incharges) or b) …on you as a client (clients)”. However, we also assured them that we were not recording their names or identifying facilities so there was no way to link their statements to them and we would not report results by facility name. The Study Information Sheet given to in charges and posted in facilities stated: “The aim of this study is to strengthen the body of programmatic learning around the integration of PPFP services into maternal, newborn, child health and nutrition services. It is a descriptive evaluation that will investigate and compare details on services for pregnant women and those who have had a child in the last 2 years and how they integrate aspects of maternal and infant young child nutrition and/or family planning in selected facilities and community programs in Bondo and Embu sub-counties in Kenya and Jharkhand and Bihar states in India. The objectives of the study are to describe implementation models, to understand barriers and facilitating factors related to the implementation and scale up of each model, and to describe client perceptions of each model.” The previous reviewer also commented on scoring for integration and waiting time and privacy. However, the methods described that analysis was in phases, with the first phase on levels of integration, phase 2 relating qualitative results to whether facilities are well integrated or not, and phase 3 the effect of these models of integration on clients experience of care. We have reorganized the results to include a clearer section on experience of care. We acknowledge the small number of respondents, which adds a degree of uncertainty around the responses regarding aspects of quality of care such as privacy and waiting times. The reviewer is correct that qualitative interviews with client may have provided richer understanding of the quantitative data. However, we have little reason to doubt clients who expressed concerns about the services they received. As the reviewer pointed out, courtesy bias would suggest under-reporting of unsatisfactory or nuisances during a health visit. The concerns about the presentation of methods are similar to the previous reviewer’s. We have sought to address this by adding a text box to further describe the methodology, including the specific questions which were used to assess levels of integration. As to why only 13 facilities have integration scores, the reviewer likely missed a sentence under the Data Management and analysis section (paragraph with heading “FP integration and qualitative data analysis” in bold) which states: “We excluded two facilities from Kenya where we did not collect client flow data because they had fewer than five client respondents per service area.” In addition, Table 2 notations point to two facilities labeled KO03 and KO06 who are excluded and the reasons why are stated. We agree that re-organizing the Results section to reflect the phases of our analysis makes sense. Thank you for the suggestion. We appreciate the concerns about the small numbers of clients for the client experience phase of the analysis. However, we choose to retain the data on client experiences since our study did not include qualitative interviews with clients to obtain their perspectives, and those perspectives are important. We added a statement to introduce the discussion which seeks to frame the implications for policymakers." } ] }, { "id": "51874", "date": "27 Aug 2019", "name": "Ilene S. Speizer", "expertise": [ "Reviewer Expertise Evaluation researcher focusing on family planning programs" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper examines integration levels of MNCH and FP services in hospitals in two states in India and a hospital and health centers in two counties in Kenya. The strength of the paper is the use of multiple data sources (qualitative and quantitative) to inform the analysis on an important topic. A limitation of the paper is the small sample of facilities in each country which means it is not possible to determine if some facilities (e.g., hospitals or health centers) are more amenable to integration than others. Related to this is that the selection of facilities suggests a preference for higher performing (on integration) facilities and thus, even though not all the included facilities were highly integrated, the results cannot be generalized across other similar types of facilities. An additional limitation includes that it is not possible to distinguish whether the findings are indicative of higher/lower integration and/or higher/lower quality of services. Quality discussions are notably absent from the paper and might be worth including since integration is a component of higher quality services. Below I highlight a few other comments:\n\nThe authors mention the India government initiative to have dedicated PPFP counselors in health facilities. The authors fail to note if the included India facilities have a dedicated counselor or not and if this is correlated with the level of integration observed in this study.\n\nMore detail is needed on the measurement of integration. The description of “as done in other studies” is vague and not informative enough for this paper.\n\nTable 1 – the median age of participants is provided – is this for clients or for all study participants including providers and key informant interviews? I expect this is just for clients and thus this should be clarified.\n\nTable 2 – you talk about “dark gray” but in the formatted version of the table, it is not clear what is “dark gray.” Also, do you need a “facility overall” column that is inferred in the text?\n\nFor the quotes, it would be useful to know if the respondent is in a high, medium or low integrated facility. This is included with some but not all the quotes.\n\nThe text starts with “All MNCH clients” when it is discussing Table 3, however, Table 3 only includes ANC clients. Make sure this is clear in the text (and possibly explain why only the ANC clients are shown).\n\nThe consistent and affordable supply of FP and the provider bias sections are particularly relevant to the quality of care issue mentioned above. Quality could be raised in the introduction to help tie integration to quality and the issues in this section.\n\nThe explanation of the inconsistent results in India relate to client volume (which makes sense), however, this is the first time that client volume is mentioned in the paper. Given that all facilities in India are hospitals, I wondered how client volume plays into the results. Can client volume be discussed earlier. Also, are some of these hospitals private and others public and does that affect integration (and quality)? For Kenya, it explicitly says that public facilities were included but this detail was not found for India.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5119", "date": "13 Jan 2020", "name": "Anne Pfitzer", "role": "Author Response", "response": "Thank you for including an assessment of strengths and limitations in your review. We acknowledge the limitation of a small number of facilities in each country. Also, we selected facilities that received programmatic support, thus we can be very clear that finding on integration are not generalizable. In fact, we believe our findings suggest that programs to encourage integration take hold unevenly across facilities, and we have sought to better understand how to encourage greater effective integration of services. We respectfully dispute your assertion that integration is a component of higher quality services. We looked at traditional quality measures across levels of integration (and in fact there may be a tradeoff in quality with greater integration if not careful), but considered integration a distinct aspect of service delivery. That being said, we added a sentence to the introduction highlighting parallels between integration and quality. We would have liked to respond more specifically to the suggestion about clarifying whether all study facilities had a counsellor. However, we did not collect this information. We can report that we had transcripts of qualitative interviews with counsellors in 4 out of the 6 Indian hospitals. The comment about detail on measurement of integration is consistent with other reviewers. Our response is also the same. We decided that it is not appropriate to include a note about the median age of clients in Table 1 and removed it. The suggestion about detailing the respondents facilities’ level of integration for the qualitative quotes echoes the 2nd reviewer. We have added this. Thank you for the keen observations about inconsistencies. Specifically, About the legend of Table 2 – the text was not amended when we changed the formatting of the table. This has been corrected. Discrepancies between the text and Table 3. All MNCH clients were asked that question, but we only report on ANC clients because of small numbers of PNC and child health clients (indicated in methods section). We have revised. We added a statement about client volume in the sample description of the methods section. The lack of precision about public vs private settings. We have added a clarification that all facilities in this study were public in the sampling section of the methods." } ] } ]	1	https://f1000research.com/articles/8-229
https://f1000research.com/articles/9-15/v1	13 Jan 20	{ "type": "Correspondence", "title": "Detection of compound heterozygous variants in LPIN1 does not necessarily imply pathogenicity in a patient with rhabdomyolysis", "authors": [ "Josef Finsterer", "Rahim Aliyev", "Rahim Aliyev" ], "abstract": "In a recent article by Yim et al., a 15-month-old male is described who experienced severe rhabdomyolysis with a creatine-kinase value (CKV) of 127494 U/l one day after intramuscular injection of an unidentified drug by the general practitioner. Rhabdomyolysis was not attributed to this injected drug but to compound heterozygous variants in LPIN1. The study has a number of shortcomings. Triggers of rhabdomyolysis should be unequivocally identified, a more extensive family history should be taken, and previous CKVs should be provided. Functional and biochemical tests should be carried out to confirm or exclude pathogenicity of the LPIN1 variants.", "keywords": [ "LPIN1", "rhabdomyolysis", "myoglobinurea", "renal insufficiency", "genetics" ], "content": "Correspondence\n\nIn a recent article, Yim et al. reported a 15-month-old male who experienced severe rhabdomyolysis with a creatine-kinase value (CKV) of 127494 U/l one day after intramuscular injection of an unidentified drug by the general practitioner (GP)1. Rhabdomyolysis was not attributed to this injection but to compound heterozygous variants in LPIN11. Each of the parents carried one of these variants but was clinically unaffected1. This correspondence article provides reasonings as to why the detection of heterozygous variants in LPIN1 does not necessarily imply pathogenicity in this case.\n\nRhabdomyolysis may not only occur in mitochondrial disorders (beta-oxidation defects are mitochondrial disorders) and glycogenoses, but more frequently in response to drugs or toxins2. Thus, it is crucial to find out which drug the GP injected one day prior to admission, not only to identify the compound, but also to ensure that other patients were not exposed to any hazardous risks due to a possibly toxic drug.\n\nSince the variant c.1949_1967dupGTGTCACCACGCAGTACCA was classified as likely pathogenic, the variant c.2410G>C as a variant of unknown significance, and since one parent each carried one of either variants, it is conceivable that the parent who carried the variant c.1949_1967dupGTGTCACCACGCAGTACCA also had experienced muscle manifestations previously. However, the family history is described as negative for rhabdomyolysis, malignant hyperthermia, or neuromuscular disorders making the pathogenicity of this variant quite unlikely. However, an extended family history should also be taken from the grandparents from the mother’s and father’s side to assess if they ever experienced any muscle symptoms.\n\nSince LPIN1 variants have been previously reported in association with recurrent rhabdomyolysis3,4, it is quite likely that the index patient or one of the parents had elevated CKVs. Thus, the records of the index patient or the parent who carried the likely pathogenic LPIN1 variant should be checked to see if these individuals ever showed elevated CKVs. Of particular interest are CKVs after birth, exercise, infection, anaesthesia, or application of drugs. This is because CKV may particularly increase with these conditions. Since the younger sister of the index patient also carried the compound heterozygous LPIN1 variants, we should be informed about the course of the mother’s pregnancy with this younger child, about the sister’s CKVs at birth and later, and further follow-up, including genetic counselling.\n\nIn the case report1, the patient has a second episode of rhabdomyolysis at the age of six years. Current medication the index patient was taking at the time of this second episode should be examined, and also if the cause could have been triggered by exercise5. A male of 6 years of age most likely is lively and usually highly physically active.\n\nOverall, this interesting case report has some limitations, which should be addressed before attributing rhabdomyolysis to the LPIN1 variants. Triggers of rhabdomyolysis should be unequivocally identified, a more extensive family history taken, and previous CKVs provided. Functional and biochemical tests should be carried out to confirm or exclude pathogenicity attributed to LPIN1 variants.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "References\n\nYim SW, Chan TYC, Belaramani KM, et al.: Case Report: The first probable Hong Kong Chinese case of LPIN1-related acute recurrent rhabdomyolysis in a boy with two novel variants [version 1; peer review: 2 approved]. F1000Res. 2019; 8: 1566. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCervellin G, Comelli I, Benatti M, et al.: Non-traumatic rhabdomyolysis: Background, laboratory features, and acute clinical management. Clin Biochem. 2017; 50(12): 656–662. PubMed Abstract \| Publisher Full Text\n\nMeijer IA, Sasarman F, Maftei C, et al.: LPIN1 deficiency with severe recurrent rhabdomyolysis and persistent elevation of creatine kinase levels due to chromosome 2 maternal isodisomy. Mol Genet Metab Rep. 2015; 5: 85–88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBergounioux J, Brassier A, Rambaud C, et al.: Fatal rhabdomyolysis in 2 children with LPIN1 mutations. J Pediatr. 2012; 160(6): 1052–4. PubMed Abstract \| Publisher Full Text\n\nRausa J, Shetty I, Loomba RS: Troponin elevation in the setting of exercise-induced rhabdomyolysis in an athletic teenager. Cardiol Young. 2019; 3: 1–4. PubMed Abstract \| Publisher Full Text" }	[ { "id": "68520", "date": "06 Aug 2020", "name": "Panupong Hansrivijit", "expertise": [ "Reviewer Expertise Minor revision is advised without the need to return the article to me." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for pointing out other etiologies that the original authors might have missed. However, I agree with the original authors' conclusion that rhabdomyolysis in this infant is related to spontaneous rhabdomyolysis from compound heterozygous variants of LPIN1 but the fact that we do not know what medication was given has raised some concerns for the accuracy of the diagnosis. Please add another reference that covers the genetic perspectives of rhabdomyolysis as attached.1 I believe this additional article will be helpful to the readers who are interested in reading up further.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Yes\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "68525", "date": "13 Aug 2020", "name": "Karolina M Stepien", "expertise": [ "Reviewer Expertise Inherited Metabolic Diseases" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI note the comments from Finsterer and Aliyev. I agree that it would be worth learning more about the first episode of rhabdomyolysis in this child who was confirmed to be compound heterozygous for two mutations causing LIPIN1. A causal relationship between this episode and the administered injection needs to be considered, and detail is given on what the injection was including dose, route, number of doses, indication, etc. This is a fundamental point that has not been addressed in the case report. Drug-induced rhabdomyolysis is relatively common. Although 40% of heterozygous carriers for LPIN1 missense mutations may be symptomatic, myopathy has been reported in these individuals in response to statin drug treatment (Zhang et al. 2014)1. The authors listed several factors that may trigger rhabdomyolysis in LIPIN1. The fact that in humans episodes of myoglobinuria in LIPIN1 are mostly precipitated by febrile illnesses (Michot et al. 2014)2 emphasizes a critical role of the inflammatory stress response as a potential triggering factor of rhabdomyolysis. I agree with the authors that there are some limitations of the case report by Yim et al. 2019. Firstly, a CK of 127,000 U/L may have detrimental consequences in a toddler. Dexamethasone was shown to decrease the number of lipid droplets in lipin‐1 deficient patients' myoblasts with a decrease in peak CK concentration during acute rhabdomyolysis. This therapy may prove to be beneficial for severe decompensation (Maijer et al. 2015)3. Was it considered during the acute illness? What about renal function and fluid management?\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Yes\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "68527", "date": "17 Aug 2020", "name": "Pushpa Raj Joshi", "expertise": [ "Reviewer Expertise Meuromuscular disordres", "mitochondrial myopathy", "Rhabdomyolysis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe correspondence ‘Detection of compound heterozygous variants in LPIN1 does not necessarily imply pathogenicity in a patient with rhabdomyolysis’ by Josef Finsterer and Rahim Aliyev well argue against the conclusion of original submission ‘Case Report: The first probable Hong Kong Chinese case of LPIN1-related acute recurrent rhabdomyolysis in a boy with two novel variants’ by SW Yim and colleagues. In this submission, the authors are not sure which drug was administered to the patient prior to admission and Dr. Finisterer and Dr. Aliyev rightly point out that Rhabdomyolysis should not have necessarily be triggered by the LPIN1 mutations but might have been due to the effect of the compounds in the injection. SW Yim and colleagues should find out the composition of the injection and prove that rhabdomyolysis was not triggered by any of the compounds in the injection. On the other hand, it is very likely that the conclusion of SW Yim might be correct. Although the pathogenicity of the reported mutations is not clear, the compound heterozygosity might be the cause of rhabdomyolysis and the mutations are, in fact, pathogenic. This is strongly supported by the fact that the child suffered from the second attack of rhabdomyolysis. However, the report on detailed family history, as pointed out by Dr. Finisterer and Dr. Aliyev, will be helpful in arguments about the pathogenicity of the mutations. Despite autosomal recessive inheritance of LPIN1 mutations, the parent(s) with only one mutation might also suffer from attack(s) of rhabdomyolysis with very mild to severe intensity. Symptomatic heterozygous cases are sporadically reported in other autosomal recessive disorders such as CPT II deficiency, also contributing to rhabdomyolysis. Overall, the original submission is interesting but needs to address the points raised by Dr. Finisterer and Dr. Aliyev to draw the conclusion that rhabdomyolysis is triggered by novel LPIN1 mutations.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Yes\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "68521", "date": "28 Aug 2020", "name": "Chiara Pizzamiglio", "expertise": [ "Reviewer Expertise Inherited muscular disorders", "mitochondrial diseases", "rhabdomyolysis." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have read with interest the correspondence from Josef Finsterer and Rahim Aliyev entitled “Detection of compound heterozygous variants in LPIN1 does not necessarily imply pathogenicity in a patient with rhabdomyolysis”.\n\nI agree that in the article by Yim et al. the triggers of rhabdomyolysis have not been clearly determined and discussed. More importantly, the drug that was injected before the first episode of rhabdomyolysis was not specified so it is not possible to exclude that it triggered the episode. The identification of the trigger is crucial as it can guide the process of the differential diagnosis (Quinlivan R and Jungbluth H, 2012).1\n\nYim et al. do not clearly state what type of genetic testing was performed in the patient, i.e. rhabdomyolysis panel, exome, or genome sequencing. This is important to understand what other genetic causes of recurrent rhabdomyolysis have been excluded in the patient, especially given the fact that a muscle biopsy was not performed.\n\nIn heterozygous carriers, LPIN1 mutation can cause cramps, myalgia and can trigger statin-induced myotoxicity, although it is not uncommon for parents to be asymptomatic. However, in the text, it is not specified if parents were taking statin.\n\nDespite the points discussed by Finsterer and Aliyev that should be addressed in the original article, the recurrent rhabdomyolysis, the fever in both episodes, the age of onset, the normal CK and examination between episodes, are in line with descriptions of LPIN1 associated rhabdomyolysis.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Yes\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-15
https://f1000research.com/articles/8-1211/v1	29 Jul 19	{ "type": "Antibody Validation Article", "title": "Validation of a commercial antibody to detect endogenous human nicastrin by immunoblot", "authors": [ "Rosana A. Mesa", "Elisha D.O. Roberson", "Rosana A. Mesa" ], "abstract": "Nicastrin (NCSTN) is a transmembrane glycoprotein that is part of the gamma-secretase complex. Gamma-secretase is a protease complex that cleaves type-I single-pass transmembrane proteins. There are many potential substrates for this complex, including NOTCH receptors and amyloid precursor proteins (APP). There are a number of commercial antibodies to nicastrin, but they do not agree on expected peptide size. We confirmed the specificity of a C-terminal binding rabbit anti-human antibody from Sigma-Aldrich (#N1660) using wildtype HEK293 cells and HEK293 cells deleted for nicastrin. The wildtype cells showed a prominent band at approximately 110 kDa. We confirmed this larger than expected sized was due to glycosylation by treating the lysate with peptide-N-glycosidase F (PNGase F), which reduced the band to less than 75 kDa. These data suggest that this polyclonal is specific for nicastrin and can detect endogenous levels of protein.", "keywords": [ "nicastrin", "gamma secretase", "polyclonal", "human", "western blot" ], "content": "Introduction\n\nThe γ-secretase complex is a multi-subunit, intramembrane protease (reviewed1). It cleaves type-I single-pass transmembrane proteins within their transmembrane domain. This can lead to the release of an intracellular and an extracellular domain that may perform other functions. Examples include the cleavage of amyloid precursor protein (APP) to produce amyloid beta and the cleavage of activated NOTCH receptors to release their intracellular domain for translocation to the nucleus2.\n\nGamma-secretase is composed of several proteins, including a presenilin protease (PSEN1 or PSEN2), the presenilin enhancer gamma-secretase subunit (PEN2), an anterior pharynx-defective 1 protein (APH1A or APH1B), and nicastrin (NCSTN)3. Nicastrin acquires extensive N-linked glycosylation during its maturation4,5. The three-dimensional structure of human gamma-secretase shows that the heavily glycosylated ectodomain of nicastrin forms a horseshoe-like clamp on the extracellular portion of the complex6,7. It is thought that NCSTN may help control substrate selectivity. There are multiple commercial antibodies for NCSTN available, but they do not agree on the expected product size. We validated one commercial polyclonal antibody (#N1660; Sigma-Aldrich) using HEK293 wildtype and nicastrin knockout cells.\n\n\nMethods\n\nWe used a commercially available rabbit anti-human IgG polyclonal antibody that targets human nicastrin (#N1660; Sigma-Aldrich, St. Louis, MO, USA; RRID:AB_477259) which is has performed well in some previous publications8,9. The antibody was raised against Uniprot nicastrin peptide Q92542 (709 amino acid total size). The polyclonal was generated by challenging rabbits with a synthetic peptide corresponding to the C-terminal cytoplasmic domain of nicastrin (peptides 693-709) fused with keyhole limpet hemocyanin as an adjuvant.\n\nThe technical documentation claims this subsequence is identical to the matching region of nicastrin in mouse. However, aligning Q92542 to the primary mouse nicastrin peptide sequence (NP_067620.3) with Clustal Omega10,11 actually shows 1 mismatch (94.1% identity; Figure 1). It’s unclear if this discrepancy is due to changes to either the human or mouse peptide sequence for the most common isoform over time as the references have been updated.\n\nShown is a partial alignment between human and mouse nicastrin. The highlighted area represents the peptides use for generation of the polyclonal antibody. Asterisks represent a matching amino acid between the two sequences, and spaces are mismatches.\n\nWe used a mouse anti-human beta actin monoclonal antibody (#AB6276; Abcam, Cambridge, MA, USA; RRID:AB_2223210) as a loading control. The details of all primary and secondary antibodies are summarized in Table 1.\n\nWe purchased the Human Embryonic Kidney cell line (HEK293) from the ATCC (CRL-1573). We cultured all cells at 37°C and 5% CO2. For culture media, we used Dulbecco’s Modified Eagle’s Media (DMEM; Gibco, Thermo-Fisher Scientific, #11965-084) supplemented with 5% Fetal Bovine Serum (FBS; Gibco, Thermo-Fisher Scientific, #26140-079), 1% HEPES (Corning, #25-060-CI), 100 U/mL penicillin / streptomycin (Gibco, Thermo-Fisher Scientific, #15140-122), and 2 mM glutamine (Corning, #25-005-CI).\n\nWe used a HEK293 NCSTN knockout line we had previously generated using CRISPR/Cas9 genome-editing12. Briefly, we synthesized our single-guide RNA as an IDT gBlock and cloned it into the pCR-Blunt TOPO vector. We co-transfected the single-guide RNA vector along with humanized Cas9 (RRID: Addgene_43861) into HEK293 cells, plated to single colonies, and screened for deleted clones by sequencing (Sequence Read Archive project PRJNA268374) and RT-qPCR (Data available from figshare, see source data13). Full methodology for RT-qPCR is provided in the supplementary material of Cao et al.12\n\nReagent details can be found in Table 2 and Table 3. We harvested cells at ≥90% confluence and pelleted them by centrifugation at 4°C and 400 ×g for 5 minutes. We washed the cell pellet three times in 10 mL of cold phosphate buffered saline (PBS). We then added 300 µL of cold lysis buffer (50 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 1.0% NP-40, and 1.5% protease inhibitor cocktail) and lysed the cells with constant agitation for 30 minutes at 4°C. We removed insoluble debris by centrifugation for 15 minutes at 4°C and 10,400 ×g. We determined the concentration of the cleared lysates using a Pierce BCA assay kit (#23227). We stored the lysates in aliquots at -80°C until further use.\n\nWe used peptide-N-glycosidase F (PNGase F; #P0704S; New England Biolabs, Ipswich, MA, USA) to remove N-linked sugars. We denatured about 50 µg of protein in glycoprotein denaturing buffer (included with NEB kit; 0.5% SDS, 40 mM DTT) at 100°C for 10 minutes, and then incubated the lysate with PNGase F for 3 hours at 37°C, according to the manufacturer’s instructions. We treated a control in parallel under the same conditions, but omitted the PNGase F enzyme.\n\nWe denatured the protein lysate by boiling for 5 minutes in Laemmli sample buffer (5% β-mercaptoethanol). We resolved the proteins on precast 7.5% polyacrylamide gels (Mini-protean TGX; Bio-Rad, Hercules, CA, USA) after loading approximately 20 µg of lysate. We used the Precision Plus Dual-Color Standard as a molecular weight marker (Bio-Rad, Hercules, CA). We prepared PVDF membranes (0.45 µm) by incubating 2 minutes in 100% isopropanol, washing in Milli-Q water for 2 minutes, and equilibrating in transfer buffer for 10 minutes. We transferred separated proteins to the PVDF membrane in transfer buffer without methanol at 200 mA for 2 hours. We blocked the membrane by incubating in blocking buffer (TBST with 5% skim milk powder) for 1 hour at room temperature with gentle rocking. We probed the membrane using primary antibodies to nicastrin (1/1000) and beta actin (1/5000) diluted in blocking buffer overnight at 4°C with gentle rocking. We removed excess unbound antibody by rinsing the membranes 5 times for 10 minutes each in TBST buffer. The anti-mouse and anti-rabbit secondary antibodies were both conjugated to horseradish peroxidase (HRP). We incubated the membranes with secondary antibody (1/7000) in blocking buffer for 1.5 hours at room temperature, followed by washing 5 times for 10 minutes each in TBST. We used the Supersignal West Pico Chemiluminescent Substrate reagent (ThermoFisher, Waltham, MA) to detect secondary antibodies.\n\n\nResults\n\nWe collected protein lysates from wildtype HEK293 cells and HEK293 NCSTN knockouts. The manufacturer provided example blots were derived from HEK293 cells, but used an overexpression construct. In wildtype HEK293 cell lysates, a single, strong band at ~110 kDa can be seen on the blot, and this band is missing in the nicastrin knockout line lysates (Figure 2A, underlying data14,15). The loading controls for the wildtype replicates and knockout replicates all show the expected band for actin (Figure 2B, underlying data14,15), supporting that the loss of the nicastrin band is specific to the knockout and not a loading error. It is worth noting that despite a low background, the nicastrin blots showed an approximately 25 kDa band in both wildtype and knockout lysates. We searched the protein sequence used to develop the antibody (KADVLFIAPREPGAVSY) with protein blast using the Homo sapiens non-redundant peptide database automatically adjusted for short queries, but only matches to nicastrin had a reasonable e-value (2×10-9 to 7×10-11). It is therefore unclear if this band is from a non-specific contaminant in the antibody, a similar peptide that is poorly annotated in the non-redundant protein database, or a nicastrin degradation product.\n\nA. The NCSTN antibody binds to endogenous levels of protein in wildtype (WT) HEK293 cells with a band at ~110 kDa. The band is absent in NCSTN knockout (KO) cells. Both replicates show an unidentified band at 25 kDa. B. The actin antibody shows the expected ~42 kDa band in both replicates of wildtype and knockout cells. Abbreviations: rep., replicate.\n\nThe nicastrin antibody documentation lists the expected fragment size as approximately 110 kDa, and this band size was confirmed on our blots. However, calculating the fragment size of human nicastrin protein sequence Q92542 using Expasy tools16 gives an estimated 78.4 kDa size for the nascent fragment and a reduced 75.2 kDa size after cleavage of the signal peptide. We hypothesized this discrepancy might be due to glycosylation.\n\nWe tested this hypothesis by first treating the lysates PNGase F, which will release asparagine-linked oligosaccharides. This reduced the molecular weight of the nicastrin band to less than 75 kDa (Figure 3A, underlying data17,18) without affecting the actin band (Figure 3B underlying data17,18). This phenomenon of a smaller than expected nicastrin band has been observed previously19. It is possible that a longer signal sequence than expected is cleaved from the nascent peptide, or the charge profile of the polypeptide affects its migration.\n\nA. In lysates untreated with PNGase F (-), the expected ~110 kDa band is present. With PNGase F treatment (+), the band regresses to less than 75 kDa. B. In both PNGase treated and untreated lysates, the beta actin band is unchanged.\n\n\nConclusion\n\nWe tested by immunoblot an anti-nicastrin antibody using HEK293 cell lysates. Our results show that the antibody is sensitive enough to detect endogenous protein with reasonable specificity. It is able to bind to both glycosylated nicastrin and nicastrin without sugar linkages. It is unclear how well the antibody would work for cell staining due to the non-specific 25 kDa band we observed on nicastrin blots. Based on these data obtained with the protocols described above, we can confirm the utility of this nicastrin antibody for immunoblotting.\n\n\nData availability\n\nHome sapiens HEK293 NCSTN knockout by Cas9, Accession number: PRJNA268374\n\nFigshare: HEK293 nicastrin knockout RT-qPCR. https://doi.org/10.6084/m9.figshare.7578539.v113\n\nThis project contains the following source data:\n\nknockout_rtqpcr.csv (Raw Ct values of RT-qPCR confirming the knockout (CRISPR-Cas9 mediated) of nicastrin in HEK293 cells.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: NCSTN antibody validation - actin antibody in HEK293 knockout line. https://doi.org/10.6084/m9.figshare.8952968.v116\n\nThis project contains the following underlying data:\n\nAntibodyValidation_NCSTN_KO_actin_Ab.svg (TIF image of actin antibody blot stored in a scaleable vector graphic file)\n\nFigshare: NCSTN antibody validation - NCSTN antibody in HEK293 knockout line. https://doi.org/10.6084/m9.figshare.8952953.v117\n\nThis project contains the following underlying data:\n\nAntibodyValidation_NCSTN_KO_NCSTN_Ab.svg (TIF image of NCSTN antibody blot stored in a scaleable vector graphic file)\n\nFigshare: NCSTN antibody validation - actin antibody in HEK293 knockout line after PNGase treatment. https://doi.org/10.6084/m9.figshare.8952983.v118\n\nThis project contains the following underlying data:\n\nAntibodyValidation_NCSTN_PNGase_actin_Ab.svg (TIF image of actin antibody blot stored in a scaleable vector graphic file)\n\nFigshare: NCSTN antibody validation - NCSTN antibody in HEK293 knockout line after PNGase treatment. https://doi.org/10.6084/m9.figshare.8952977.v119\n\nThis project contains the following underlying data:\n\nAntibodyValidation_NCSTN_PNGase_NCSTN_Ab.svg (TIF image of NCSTN antibody blot stored in a scaleable vector graphic file)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThis work was partially supported by the Washington University in St. Louis (WUSTL) Rheumatic Diseases Research Resource-based Center (RDRRC) [P30-AR073752] and the Washington University Institute for Clinical and Translational Sciences [UL1-TR000448].\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Professor Matthias Voss (Kiel University, Kiel, Germany) and Professor Yasuomi Urano (Doshisha University, Kyoto, Japan) for suggestions and sharing protocols.\n\n\nReferences\n\nWolfe MS: Structure and Function of the γ-Secretase Complex. Biochemistry. 2019; 58(27): 2953–2966. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYu G, Nishimura M, Arawaka S, et al.: Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and betaAPP processing. Nature. 2000; 407(6800): 48–54. PubMed Abstract \| Publisher Full Text\n\nKimberly WT, LaVoie MJ, Ostaszewski BL, et al.: Gamma-secretase is a membrane protein complex comprised of presenilin, nicastrin, Aph-1, and Pen-2. Proc Natl Acad Sci U S A. 2003; 100(11): 6382–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang DS, Tandon A, Chen F, et al.: Mature glycosylation and trafficking of nicastrin modulate its binding to presenilins. J Biol Chem. 2002; 277(31): 28135–42. PubMed Abstract \| Publisher Full Text\n\nKimberly WT, LaVoie MJ, Ostaszewski BL, et al.: Complex N-linked glycosylated nicastrin associates with active gamma-secretase and undergoes tight cellular regulation. J Biol Chem. 2002; 277(38): 35113–7. PubMed Abstract \| Publisher Full Text\n\nLu P, Bai XC, Ma D, et al.: Three-dimensional structure of human γ-secretase. Nature. 2014; 512(7513): 166–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBai XC, Yan C, Yang G, et al.: An atomic structure of human γ-secretase. Nature. 2015; 525(7568): 212–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVoss M, Kunzel U, Higel F, et al.: Shedding of glycan-modifying enzymes by signal peptide peptidase-like 3 (SPPL3) regulates cellular N-glycosylation. EMBO J. 2014; 33(24): 2890–905. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKotani R, Urano Y, Sugimoto H, et al.: Decrease of amyloid-β levels by curcumin derivative via modulation of amyloid-β protein precursor trafficking. J Alzheimers Dis. 2017; 56(2): 529–42. PubMed Abstract \| Publisher Full Text\n\nMadeira F, Madhusoodanan N, Lee J, et al.: Using EMBL-EBI Services via Web Interface and Programmatically via Web Services. Curr Protoc Bioinformatics. 2019; 66(1): e74. PubMed Abstract \| Publisher Full Text\n\nSievers F, Wilm A, Dineen D, et al.: Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol. 2011; 7: 539. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCao L, Morales-Heil DJ, Roberson EDO: Nicastrin haploinsufficiency alters expression of type I interferon-stimulated genes: the relationship to familial hidradenitis suppurativa. Clin Exp Dermatol. 2019; 44(4): e118–e25. PubMed Abstract \| Publisher Full Text\n\nRoberson E, Morales-Heil D, Cao L: HEK293 nicastrin knockout RT-qPCR [Internet]. FigShare. 2019. Reference Source\n\nRoberson E, Mesa R: NCSTN antibody validation - actin antibody in HEK293 knockout line. figshare. Figure. 2019. http://www.doi.org/10.6084/m9.figshare.8952968.v1\n\nRoberson E, Mesa R: NCSTN antibody validation - NCSTN antibody in HEK293 knockout line. figshare. Figure. 2019. http://www.doi.org/10.6084/m9.figshare.8952953.v1\n\nArtimo P, Jonnalagedda M, Arnold K, et al.: ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res. 2012; 40(Web Server issue): W597–603. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoberson E, Mesa R: NCSTN antibody validation - actin antibody in HEK293 knockout line after PNGase treatment. figshare. Figure. 2019. http://www.doi.org/10.6084/m9.figshare.8952983.v1\n\nRoberson E, Mesa R: NCSTN antibody validation - NCSTN antibody in HEK293 knockout line after PNGase treatment. figshare. Figure. 2019.\n\nLee SH, Sharma M, Südhof TC, et al.: Synaptic function of nicastrin in hippocampal neurons. Proc Natl Acad Sci U S A. 2014; 111(24): 8973–8. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "51767", "date": "06 Aug 2019", "name": "Satoru Funamoto", "expertise": [ "Reviewer Expertise Neuropathology and biochemistry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is very important to validate commercial antibodies. However this reviewer thinks it would help to enhance the value of this manuscript if authors consider the below:\nSigma’s anti-Nicastrin antibody has been already a golden standard for Nicastrin detection. It is not clear how other commercial antibodies do not agree on size. It is very important to show bands detected with several commercial anti-Nicastrin antibodies. This could be very informative.\n\nIt is already published that PNGase and Endo H treatments altered migration distance of Nicastrin in gels1.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nAre sufficient details of materials, methods and analysis provided to allow replication by others? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5142", "date": "10 Jan 2020", "name": "Elisha Roberson", "role": "Author Response", "response": "Reviewer question 1: Sigma’s anti-Nicastrin antibody has been already a golden standard for Nicastrin detection. It is not clear how other commercial antibodies do not agree on size. It is very important to show bands detected with several commercial anti-Nicastrin antibodies. This could be very informative. Response: We agree that a comprehensive screening of multiple antibodies would be fruitful. For this experiment we focused on validating a commonly used antibody to ensure that it was suitable for our future experimental designs. We are considering this project in the future, given that we can generate the necessary funds to purchase the requisite antibodies. Reviewer question 2: It is already published that PNGase and Endo H treatments altered migration distance of Nicastrin in gels. Response: It has been previously published in your suggested reference from 2003. We are able to confirm that this effect still persists, and that >15 years later the antibody specificity is still acceptable. We appreciate the suggestion for this reference and have appropriately cited it in the revised version." } ] }, { "id": "51771", "date": "18 Sep 2019", "name": "Lucia Chavez-Gutierrez", "expertise": [ "Reviewer Expertise Biochemistry", "Intramembrane proteolysis", "Alzheimer's Disease" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors validated a commercially available rabbit polyclonal anti-Nicastrin antibody from Sigma-Aldrich (#N1660) in immunoblot analysis, using as inputs total lysates from wildtype and Nicastrin knockout HEK293 cell lines. The N1660 antibody, generated against a synthetic peptide corresponding to the C-terminal cytoplasmic part of human Nicastrin (693-709), identified a band at ~110 kDa only in the wild type lysate and another band at ~ 25 kDa in both wildtype and knockout lysates. The data does not clarify whether the low-molecular weight signal is a non-specific band or a nicastrin-derived peptide.\nTreatment with PNGnase F to remove all N-linked oligosaccharide chains, reduced the molecular weight of the nicastrin band to less than 75 kDa, without affecting the loading control actin band. The observed shift in molecular weight is supported by previous observations (Herreman A. et al., 20031) and the authors should acknowledge this study.\nOther Points:\nThe authors should test the N1660 antibody against mouse Nicastrin.\n\n“It is thought that NCSTN may help control substrate selectivity.” The ‘substrate receptor” model (Shah et al, 20052) has been challenged by independent studies (Chávez‐Gutiérrez et al, 20083; Zhao et al, 20104), and it is not supported by available high resolution structural data for the gamma secretase complex (Bai et al, 20155). More recent studies indicate that the ectodomain of NCT has both passive and active roles in gamma secretase-mediated proteolysis. On one hand, it restricts the access of substrates presenting large ectodomains by steric hindrance (Bolduc et al, 20166). On the other, it has an active role in the regulation of the sequential proteolysis of APP and response to gamma secretase modulators (GSMs); thus, nicastrin modulates Aβ length.\n\n“This phenomenon of a smaller than expected nicastrin band has been observed previously. It is possible that a longer signal sequence than expected is cleaved from the nascent peptide, or the charge profile of the polypeptide affects its migration.” Shah et al., 20052 provides information about the precise signal peptide sequence cleaved from the nascent NCT polypeptide.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nAre sufficient details of materials, methods and analysis provided to allow replication by others? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5143", "date": "10 Jan 2020", "name": "Elisha Roberson", "role": "Author Response", "response": "Reviewer question 1: Treatment with PNGnase F to remove all N-linked oligosaccharide chains, reduced the molecular weight of the nicastrin band to less than 75 kDa, without affecting the loading control actin band. The observed shift in molecular weight is supported by previous observations (Herreman A. et al., 2003) and the authors should acknowledge this study. Response: This is a valid point, and have cited the appropriate study. Thanks for the suggestion. Reviewer question 2: The authors should test the N1660 antibody against mouse Nicastrin. Response: As requested we used murine protein to test the antibody. This data has been added as Figure 4, and the expected band size was observed, along with the non-specific small molecular weight band that show up in the HEK293 blots. Reviewer question 3: \"It is thought that NCSTN may help control substrate selectivity.” The ‘substrate receptor” model (Shah et al, 2005) has been challenged by independent studies (Chávez‐Gutiérrez et al, 2008; Zhao et al, 2010), and it is not supported by available high resolution structural data for the gamma secretase complex (Bai et al, 2015). More recent studies indicate that the ectodomain of NCT has both passive and active roles in gamma secretase-mediated proteolysis. On one hand, it restricts the access of substrates presenting large ectodomains by steric hindrance (Bolduc et al, 2016). On the other, it has an active role in the regulation of the sequential proteolysis of APP and response to gamma secretase modulators (GSMs); thus, nicastrin modulates Aβ length. Response: These are all excellent points. It is, of course, not possible to exhaustively discuss all the possibilities for nicastrin function in gamma-secretase in the introduction, as that is more suitable to a full literature review. However, these are points worth at least mentioning, and good references to support the paper. We have revised the paper to point to some of these different evidence sets regarding nicastrin function and have added the suggested references. Reviewer question 4: “This phenomenon of a smaller than expected nicastrin band has been observed previously. It is possible that a longer signal sequence than expected is cleaved from the nascent peptide, or the charge profile of the polypeptide affects its migration.” Shah et al., 2005 provides information about the precise signal peptide sequence cleaved from the nascent NCT polypeptide. Response: Given the work from Shah determining the signal peptide sequence, we have revised this section to point out this information (referencing the paper). We have also reworded to suggest that perhaps the disparity is more likely to be due to conformation / charge profile than additional signal peptide sequence cleavage." } ] } ]	1	https://f1000research.com/articles/8-1211
https://f1000research.com/articles/9-14/v1	10 Jan 20	{ "type": "Software Tool Article", "title": "Pharmosome: an integrative and collective database for exploration and analysis of single nucleotide polymorphisms associated with disease", "authors": [ "Peter T. Habib", "Alsamman M. Alsamman", "Sameh E. Hassanein", "Kerolos M. Yousef", "Aladdin Hamwieh", "Alsamman M. Alsamman", "Sameh E. Hassanein", "Kerolos M. Yousef", "Aladdin Hamwieh" ], "abstract": "Current single nucleotide polymorphism (SNP) databases are limited to a narrow set of SNPs, which has led to a lack of interactivity between different databases, limited tools to analyze and manipulate the already existing data, and complexity in the graphical user interface. Here we introduce Pharmosome, a web-based, user-friendly and collective database for more than 30,000 human disease-related SNPs, with dynamic pipelines to explore SNPs associated with disease development, drug response and the pathways shared between different genes related to these SNPs. Pharmosome implements several tools to design primers to detect SNPs in large genomes and facilitates analysis of different SNPs to determine relationships between them by aligning sequences, constructing phylogenetic trees, and providing consensus sequences illustrating the connections between SNPs. Pharmosome was written in the Python programming language using the Django web framework in combination with HTML, CSS, and JavaScript to receive user inputs, and process and export the sorted result to the interface. Pharmosome is available from: https://pharmosome.herokuapp.com/.", "keywords": [ "SNP", "Disease", "Python", "Bioinformatics" ], "content": "Introduction\n\nWith the extending and deciphering of genomic data produced by sequencing technologies and the Human Genome Project1, much information has been discovered, such as exons, introns, domains, coding sequence, and non-coding sequences. Mutated signals have attracted tremendous attention because of their crucial impact in altering gene expression, in particular, single nucleotide polymorphisms (SNPs)2, which also act as gene molecular-markers for an associated trait. Additionally, the existence of a specific SNP can indicate precisely what disease is foreseen and the possible drugs to treat it, which is considered the ultimate goal of pharmacogenomics.\n\nPharmacogenetics, or its inclusive version pharmacogenomics (since it covers proteomic, genomic, epigenomic and transcriptomic effects on disease and drug response), has produced a vast body of research since 19973, due to its key importance in personalized medicine through investigating how far genetic variations (e.g. SNPs) are involved in disease development and determining drug targets. Thereby the safety and efficacy of an individualized drug therapy can be improved. Since the relationships between molecular data pertaining to patients and their disease phenotype are complex and difficult to determine manually, scientists have begun to develop and enrich the bioinformatics knowledge base with more sophisticated and accurate molecular tools to detect genetic variations for example, SNPector is a recent tool developed by the authors to detect SNP effect in drug response and disease development4. This will allow interpretation of how these tiny variations may cause direct errors, e.g. X-SNP is a common SNP type that gives rise to premature termination codons that halt gene expression5. Some SNP types involved in alteration of the protein production process cause disease6, while other regulatory SNPs7 may disrupt pathways causing cascade errors that lead to collapse of a whole pathway thereby causing disease development.\n\nElucidations about SNPs play a central role in providing recommendations for practicing physicians. In addition, a wide range of research fields have arisen out of pharmacogenomics, such as vaccinomics, which study aberrant immune responses to vaccines based on genetic makeup. For example, a specific SNP in the TLR3 gene was found to be responsible for the reduction of humoral immune responses and cell-mediated immunity to the measles vaccine8. Nutrigenomics is the science of gene–nutrient interactions, which involves research methods and clinical implementation to detect and treat nutrient-related diseases. One of the best known examples of nutrigenomics is lactase persistence, in which the gene encoding lactase is expressed past weaning. Lactase persistence in Europeans is caused by a polymorphism called “C-13910T” in the lactase phlorizin hydrolase gene promoter9, and the lack of C-1390T in many adults can lead to severe gastrointestinal discomfort and diarrhea resulting from ingesting milk due to the inability to metabolize lactose10.\n\nHere we introduce Pharmosome, a web-based, user-friendly and collective database for more than 30,000 human disease-related SNPs, with dynamic pipelines to explore SNPs associated with disease development, drug response and the pathways shared between different genes related to these SNPs. Pharmosome implements several tools to design primers to detect SNPs in large genomes and facilitates analysis of different SNPs to determine relationships between them by aligning sequences, constructing phylogenetic trees, and providing consensus sequences illustrating the connections between SNPs.\n\n\nMethods\n\nWe collected SNP-related data (e.g. SNP ID, annotation, pathway and phenotypes) from PharmGKB11, NCBI12,13, Ensemble14, DiseaseEnhancer15, GeneCards16 and Reactome17 in tab-delimited format in order to link between different available information. The collected data were categorized into four main sub-databases: SNP, Gene, Chemical and Disease. The Python 3+ programming language was utilized to read, select, filter and sort the data and to link the Python scripts with HTML and JavaScript codes.\n\nData collection. About 50% of the data was downloaded from PharmGKB, which is considered the most common database for SNP annotation. The data comprises the associated phenotype, the clinical perspectives and, considering storage space limitations, the remaining data are imported on-demand to use later by a set of Python functions we built to get access to the Application Programming Interface (API) of different databases. These other databases include GeneCards, DiseaseEnhancer, Ensembl and Reactome; thereby a user can return specific data using preset IDs and then export this information to the HTML interface. The number of data entries collected by Pharmosome is shown in Table 1.\n\nData fetching and exporting. We constructed a Python module containing 12 functions. These functions connect in different ways to import the data from the databases above either from tab-delimited files or using APIs. User-requested information is extracted and exported to the Pharmosome web interface. The Django web framework was used to build the Pharmosome web interface with HTML programming language. We used Django to build functions that receive the user input from the Pharmosome interface, process requests using a Python script, and finely export the result to the interface.\n\n\n\nGoogle Chrome browser is recommended to use the Pharmosome web interface (https://pharmosome.herokuapp.com/), but other internet browsers can also be used.\n\nSNP sub-database. In the SNP sub-database, users can enter the ID of a SNP (e.g. rs141033578) and receive output data about its related gene, chromosomal location, gene bands, summary of the normal function of the gene, the gene part responsible for enhancing the disease occurrence, pathway of defective gene (if available), gene transcripts and different splicing variants. Pharmosome also provides information that can be used to retrieve data of recent studies (specifically, SNP reference nucleotide and alternative nucleotide data and an explanation of how the SNP contributes to disease development and drug response). For example, an SNP in GSTP1 was associated with overall survival in 107 patients with metastatic colorectal cancer who received 5-FU/oxaliplatin combination chemotherapy that caused the replacement of isoleucine with valine at amino acid position 105 of the protein, which is known to substantially diminish enzyme activity18.\n\nDisease sub-database. The Disease sub-database allows users to search for disease data collected from KEGG Disease and PharmGKB databases by entering the disease name. The search result is a list of genes responsible for the disease, the ID of the SNP occurring in this gene, a description of clinical annotations related to this gene and the gene-specific chemical used in the treatment. The sub-database can be used, for example, to identify the SNP, gene and chemical related to coronary artery disease caused by a high frequency of a particular polymorphism in the PLA2 gene. This gene encodes glycoprotein IIIa, which is associated with a high prevalence of premature myocardial infarction19,20\n\nChemical sub-database. The Chemical sub-database has data from four sources: KEGG, PharmGKB, DrugBank and ChemSpider. After users enter a chemical name, the output is a list of the chemical name, trend and generic names, structure, description and pharmacodynamics. For example, by inputting bortezomib (a proteasome inhibitor), users receive output data relating to the clinical success of bortezomib, which established the ubiquitin (Ub)+proteasome system as a key therapeutic target in multiple myeloma21,22.\n\nGene sub-database. The Gene sub-database links between different sources (NCBI GenBank, Ensemble, Reactome and DiseaseEnhancer). In particular, the DiseaseEnhancer dataset represents a new approach that determines the gene part that is responsible for enhancing occurrence of disease. NCBI provides information about gene name, location, a summary of gene function and chromosomal location. Reactome displays the pathway in which genes are involved and gives an overview description. The Gene sub-database can be used to identify the pathway, splicing variants, disease enhancing region, genomic and proteomic expression profile or even to get general information. An example of this would be looking at the SLCO1B1 gene, for which various groups tested the hypothesis of whether polymorphisms in SLCO1B1 affect pharmacokinetics and the effects of drugs in humans23.\n\nSNP collector. The SNP collector is a tool within Pharmosome that is designed to find all SNPs present on the gene, related to disease, associated with the chemical compound. Users can choose between different options to collect SNPs and their clinical annotations and chemicals related to each SNP. This tool can be used to find SNPs or other information. For example, it could be used to detect the bond work which occurs on location 118 of the mu opioid gene, which was 3 fold more vigorous than the wildtype in its interaction with b-endorphin Other regulatory-SNPs in this region of the gene can be linked to other phenotypes24.\n\nPick primer. The detection of SNP existence in a DNA sample taken from patients depends on designing an appropriate primer. Primers should be compatible with the flanking sequences of SNP. As the presence of an SNP in the genome may result in disease and affect the choice of drug, there is a need to detect the presence of SNP e.g. for early disease diagnosis. The pick primer tool within Pharmosome has the important function of designing primers to detect an SNP in the genome by retrieving the SNP sequence record from the NCBI database, locating the SNP position and designing primers 50 bases before, after and within the SNP sequence.\n\nSNP phylogeny. As discussed in previous sections, there is always some relationship between different SNPs due to the complicated interaction network between different genes. In order to determine how these SNPs are related to each other, the SNP phylogeny tool constructs a phylogenetic tree that illustrates the relationships between different SNPs25 by downloading SNP and flanking sequences and commencing multiple sequence alignment to determine how far each sequence is related to others. This function could be used clarify connections in studies, such as that of Thompson et al. that showed an association of 43 SNPs in 16 genes with the response drug of atorvastatin26.\n\nFigure 1 shows the flow of information to meet the needs of users.\n\n\nUse case\n\nPharmosome deploys seven sub-databases and tools. Our approach during the building of Pharmosome, is to achieve the easiest usage. We designed each tool and sub-database to receive the user input with minimum required parameters (as shown in Table 2). Users enter the target input they require and the Pharmosome interface will automatically redirect to another page that shows the user the output results. Figure 2–Figure 5 show output on the Pharmosome web interface.\n\nThe results consist of list of drop-down menus. Each menu describes disease annotation and the associated gene to the disease.\n\nThe results consists of a list of drop-down menus. Each menu describes Drug/Chemical annotations.\n\nThe results consist of a navigation bar, and each button expands to a different annotation.\n\nThe results consists of a list of SNPs located on this gene.\n\n\nSummary\n\nIn this study, we introduce Pharmosome, an integrative and collective database for exploring and analysing human SNPs and the associated disease and drug response. Our tool deploys various functions to determine the relationships between different SNPs, construct the consensus sequence between different SNPs and to determine the pathways shared between different genes. Pharmosome also includes sub-databases to simplify, link and display data about gene functions, pathways, transcriptomes of genes, different splicing variants, clinical annotation, chemical structures and annotations of chemicals involved in the disease. The returned data are informative, user-friendly and easy to navigate. Pharmosome was written in Python 3.5, HTML and CSS with the implementation of Django (Python library) to design links between Python scripts and other languages.\n\n\nSoftware availability\n\nPharmosome web interface: https://pharmosome.herokuapp.com/\n\nSource code available from: https://github.com/peterhabib/Pharmosome_Web\n\nArchived source code as at time of publication: http://doi.org/10.5281/zenodo.358319127.\n\nLicense: MIT", "appendix": "References\n\nLander ES, Linton LM, Birren B, et al.: Initial sequencing and analysis of the human genome. Nature. 2001; 409(6822): 860–921. PubMed Abstract \| Publisher Full Text\n\nVignal A, Milan D, SanCristobal M, et al.: A review on SNP and other types of molecular markers and their use in animal genetics. Genet Sel Evol. 2002; 34(3): 275–305. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGurwitz D, Pirmohamed M: Pharmacogenomics: the importance of accurate phenotypes. Pharmacogenomics. 2010; 11(4): 469–70. PubMed Abstract \| Publisher Full Text\n\nHabib PT, Alsamman AM, Hassanein SE, et al.: SNPector: SNP inspection tool for diagnosing gene pathogenicity and drug response in a naked sequence [version 1; peer review: awaiting peer review]. F1000Res. 2019; 8: 2133. Publisher Full Text\n\nSavas S, Tuzmen S, Ozcelik H: Human SNPs resulting in premature stop codons and protein truncation. Hum Genomics. 2006; 2(5): 274–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBond J, Scott S, Hampshire DJ, et al.: Protein-truncating mutations in ASPM cause variable reduction in brain size. Am J Hum Genet. 2003; 73(5): 1170–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDe Gobbi M, Viprakasit V, Hughes JR, et al.: A regulatory SNP causes a human genetic disease by creating a new transcriptional promoter. Science. 2006; 312(5777): 1215–7. PubMed Abstract \| Publisher Full Text\n\nDhiman N, Poland GA, Cunningham JM, et al.: Variations in measles vaccine-specific humoral immunity by polymorphisms in SLAM and CD46 measles virus receptors. J Allergy Clin Immunol. 2007; 120(3): 666–72. PubMed Abstract \| Publisher Full Text\n\nEnattah NS, Sahi T, Savilahti E, et al.: Identification of a variant associated with adult-type hypolactasia. Nat Genet. 2002; 30(2): 233–7. PubMed Abstract \| Publisher Full Text\n\nKaput J, Rodriguez RL: Nutritional genomics: the next frontier in the postgenomic era. Physiol Genomics. 2004; 16(2): 166–77. PubMed Abstract \| Publisher Full Text\n\nHewett M, Oliver DE, Rubin DL, et al.: PharmGKB: the Pharmacogenetics Knowledge Base. Nucleic Acids Res. 2002; 30(1): 163–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGene. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004; Accessed 2019-02-16. Reference Source\n\ndbSNP. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 1998; Accessed 2019-02-16. Reference Source\n\nKersey PJ, Allen JE, Allot A, et al.: Ensembl Genomes 2018: an integrated omics infrastructure for non-vertebrate species. Nucleic Acids Res. 2018; 46(D1): D802–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang G, Shi J, Zhu S, et al.: DiseaseEnhancer: a resource of human disease-associated enhancer catalog. Nucleic Acids Res. 2018; 46(D1): D78–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSafran M, Dalah I, Alexander J, et al.: GeneCards Version 3: the human gene integrator. Database (Oxford). 2010; 2010: baq020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFabregat A, Sidiropoulos K, Viteri G, et al.: Reactome diagram viewer: data structures and strategies to boost performance. Bioinformatics. 2018; 34(7): 1208–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStoehlmacher J, Park DJ, Zhang W, et al.: Association between glutathione S-transferase P1, T1, and M1 genetic polymorphism and survival of patients with metastatic colorectal cancer. J Natl Cancer Inst. 2002; 94(12): 936–42. PubMed Abstract \| Publisher Full Text\n\nWeiss EJ, Bray PF, Tayback M, et al.: A polymorphism of a platelet glycoprotein receptor as an inherited risk factor for coronary thrombosis. N Engl J Med. 1996; 334(17): 1090–4. PubMed Abstract \| Publisher Full Text\n\nWalter DH, Hink U, Asahara T, et al.: The in vivo bioactivity of vascular endothelial growth factor/vascular permeability factor is independent of N-linked glycosylation. Lab Invest. 1996; 74(2): 546–56. PubMed Abstract\n\nRichardson PG, Mitsiades CS, Hideshima T, et al.: Novel biological therapies for the treatment of multiple myeloma. Best Pract Res Clin Haematol. 2005; 18(4): 619–34. PubMed Abstract \| Publisher Full Text\n\nRichardson PG, Schlossman RL, Alsina M, et al.: PANORAMA 2: panobinostat in combination with bortezomib and dexamethasone in patients with relapsed and bortezomib-refractory myeloma. Blood. 2013; 122(14): 2331–7. PubMed Abstract \| Publisher Full Text\n\nKönig J, Seithel A, Gradhand U, et al.: Pharmacogenomics of human OATP transporters. Naunyn Schmiedebergs Arch Pharmacol. 2006; 372(6): 432–43. PubMed Abstract \| Publisher Full Text\n\nBond C, LaForge KS, Tian M, et al.: Single-nucleotide polymorphism in the human mu opioid receptor gene alters beta-endorphin binding and activity: possible implications for opiate addiction. Proc Natl Acad Sci U S A. 1998; 95(16): 9608–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHabib PT, Alsamman AM, Hamwieh A: BioAnalyzer: Bioinformatic software of routinely used tools for analysis of genomic data. Biotechnology. 2019; 10: 33–41. Publisher Full Text\n\nThompson JF, Man M, Johnson KJ, et al.: An association study of 43 SNPs in 16 candidate genes with atorvastatin response. Pharmacogenomics J. 2005; 5(6): 352–8. PubMed Abstract \| Publisher Full Text\n\nPeter: Pharmosome: An Integrative and Collective Database for Explorations and Analysis of SNP Associated with Disease. Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3583191" }	[ { "id": "90928", "date": "19 Aug 2021", "name": "Fuyi Li", "expertise": [ "Reviewer Expertise Bioinformatics and Computational biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study developed a novel web-based database, Pharmosome, for human disease-related SNPs. The web page of the database is well-designed. The manuscript is well written and easy to follow.\nI have several comments and suggestions:\nThe authors should provide a download page on the webserver of the database to allow the users to download the data in their database.\n\nA detailed user manual or tutorial for the webserver of Pharmosome should be provided on the webserver to guide the users to use the database.\n\nIt would be better to provide a timeline of the database, which can help to record the different versions of the database.\n\nThe authors are suggested to provide a statistics page to show the statistic summary of the database.\n\nIt would be better to have an advanced search option to allow the users to search the data with a combination of variables.\n\nThe authors should also provide an example in each search box to show the users which format of the IDs or keywords are acceptable.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "98627", "date": "18 Nov 2021", "name": "Mulin Jun Li", "expertise": [ "Reviewer Expertise Bioinformatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, the authors developed Pharmosome, a web-based, user-friendly and collective annotation database for exploring and analyzing SNPs associated with disease development, drug response and the pathways shared between different genes related to these SNPs. Pharmosome is a friendly tool to query pathogenic SNP and the relationship of disease or drug.\nHowever, this study needs some more evaluations and validations:\nMajor:\nIn the disease sub-database, Pharmosome should provide the effect of drugs on the disease and clinical/preclinical/experimental levels of evidence.\n\nIn the primer design sub-database, Pharmosome should provide specific scores to indicate the quality of primers and the feasibility of subsequent experiments.\n\nThe authors should compare Pharmosome with similar databases such as PharmGKB, PreMedKB, and CIVIC to show its specific focus and superiority.\n\nIt seems that the authors restrict the input SNPs to be located in gene regions, but the majority of disease-associating SNPs lie in the non-coding regions according to published GWAS and drug-response studies, non-coding SNPs should not be excluded therefore.\nMinor:\nThe versions of all collected resources should be provided.\n\nThere are some errors/bugs in the website, such as the hyperlink of SNP from disease page to SNP page cannot be accessed by users, please polish them accordingly.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? No\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No", "responses": [] } ]	1	https://f1000research.com/articles/9-14
https://f1000research.com/articles/8-793/v1	06 Jun 19	{ "type": "Research Article", "title": "Staffing in public health facilities after the Ebola outbreak in rural Sierra Leone: How much has changed?", "authors": [ "James Sylvester Squire", "Katrina Hann", "Olga Denisiuk", "Rony Zachariah", "Katrina Hann", "Olga Denisiuk", "Rony Zachariah" ], "abstract": "Background: The 2014-2015 Ebola outbreak in Sierra Leone led the Ministry of Health and Sanitation to set minimum standards of staffing (medical/non-medical) at the district level for the provision of basic essential health services (BPEHS). In one of the worst Ebola affected districts in Sierra Leone, we assessed staffing levels measured against these stipulated standards before, during, and 16 months after the Ebola outbreak. Methods: The study population included all health workers in 83 health facilities. We assessed staffing levels at three points in time: pre-Ebola (April 2014); the end of the outbreak (November 2015); and 16 months post-Ebola (March 2017). April 2014 was immediately prior to the Ebola outbreak and thus representative of the human resource situation before the outbreak. November 2015 was the month when Sierra Leone was declared Ebola-free, and thus reflects the end-situation after Ebola. March 2017 was two years since the launch of the BPEHS, and some progress should be expected. Results: Against recommended medical staff numbers during pre-, intra- and post-Ebola periods, deficits were 67%, 65% and 60% respectively. Similarly, against recommended non-medical staff numbers during pre-, intra- and post-Ebola periods, the deficit remained at 92% throughout. In the post-Ebola period, there was a deficit of 73% against 1,389 recommended health worker positions. Conclusions: Nothing has really changed in the state of human resources for health, and urgent measures are needed to rectify the situation and prevent a déjà vu in the advent of a new Ebola outbreak.", "keywords": [ "Outbreak response", "SORT IT", "Sustainable Development Goals", "Universal Health Coverage", "Basic Package of Essential Health Services" ], "content": "Introduction\n\nThe Ministry of Health and Sanitation of Sierra Leone has stipulated minimum staffing levels for all public health facility levels based on the Basic Package of Essential Health Services (BPEHS)1.\n\nAn observational study published in 2017 following the 2014–2015 Ebola outbreak reported alarming human resource deficits in public health facilities in Kailahun district of rural Sierra Leone2. Of 805 recommended medical staff, the deficit was 501 (62%) and hovered over 50% at all levels of health facilities. Similarly, of 569 recommended non-medical staff, the deficit was 524 (92%). The overarching message was that to meet the BPEHS1 standards, the Government would need to attract an additional 1,026 workers to Kailahun district over the period 2016–2020 (roughly 256 additional workers per annum).\n\nThree years have now passed since the end of the Ebola outbreak and the operational question is “what has changed” in terms of progress towards achieving BPEHS standards.\n\nAmong all public health facilities in Kailahun district of Sierra Leone and in relation to BPEHS standards, we thus assessed staffing levels (medical and non-medical) one month before the onset of the Ebola outbreak, during the last month of the outbreak, and 16 months thereafter.\n\n\nMethods\n\nThis was a comparative cross-sectional study using programme data. The study setting has been described before2. The study site was Kailahun district, the first district affected by the Ebola outbreak in Sierra Leone. It shares borders with the Republic of Liberia and Guinea. The health infrastructure is tiered into tertiary hospitals, district hospitals and Peripheral Health Units. The current study included all 82 functional public health facilities.\n\nThe study population included all health workers in these health facilities. We assessed staffing levels at 16 months post-Ebola (March 2017), and compared to previously reported staffing levels for pre-Ebola (April 2014) and the end of the outbreak (November 2015)1.\n\nApril 2014 was immediately prior to the Ebola outbreak and thus representative of the human resource situation before the outbreak. November 2015 was the month when Sierra Leone was declared Ebola-free, and thus representative of the end-situation after Ebola. March 2017 was selected because the revised BPEHS was launched two years prior to this date, and some progress should have been expected.\n\nData variables were sourced from the monthly district staff list (District Health Information Systems; DHIS2) and the Human Resource Management Information System. Deficits in staffing levels were derived by substracting the actual levels from the stipulated levels.\n\nEthics approval was obtained from the Sierra Leone Ethics and Scientific Review Board (dated 18 December 2018) and the Union Ethics Advisory Group (International Union against Tuberculosis and Lung Disease, Paris, France; UAG number 71/18). Since anonymized programme data were used, the requirement for informed consent was waived.\n\n\nResults\n\nTable 1 shows the medical staffing levels in relation to BPEHS standards. Of 805 recommended medical staff during the pre-Ebola and intra-Ebola periods, deficits were 539 (67%) and 528 (65%) respectively. During the post-Ebola period, a total of 815 medical staff were recommended, but the deficit was 490 (60%; a 5% improvement over the intra-Ebola period). When stratified by health facility levels, human resource gaps ranged between 31% and 71%.\n\nBPEHS: Basic Package of Essential Health Services document for improving health service delivery in Sierra Leone; CHC: Community Health Center; CHP: Community Health Post; MCHP: Maternal and Child Health Post\n\n1 Includes staff such as specialist doctors, general practitioners, clinical officers, nurses and midwives\n\n2 Pre-Ebola – April 2014; Intra-Ebola – November 2015; Post-Ebola – March 2017\n\n3 The overall recommended numbers of staff as per the BPEHS increased from 805 during the pre- and intra-Ebola period to 820 in the post-Ebola period as one new facility was added in the post-Ebola period.\n\n4 Similarly, during the post-Ebola period, 5 MCHPs were upgraded to CHPs increasing the staffing requirement for the CHPs from 240 to 265.\n\nTable 2 shows non-medical staffing levels in relation to BPEHS standards. The overall deficit remained the same at the three time-points. Of 569 recommended non-medical staff during pre- and post-Ebola, the deficits were 526 (92%) and 525 (92%), respectively. During the post-Ebola period, of 574 recommended non-medical staff, the deficit was 528 (92%).\n\nBy March 2017 and well into the post-Ebola period, a total of 1,389 health worker positions (medical and non-medical) were recommended by BPEHS, but only 371 (27%) were filled, resulting in an overall human resource deficit of 1,018 (73%).\n\nBPEHS: Basic Package of Essential Health Services document for improving health service delivery in Sierra Leone; CHC: Community Health Center; CHP: Community Health Post; MCHP: Maternal and Child Health Post\n\n1 Includes staff such as administrative staff, cleaners, cooks, maintenance workers, drivers and security personnel\n\n2 Pre-Ebola – April 2014; Intra-Ebola – November 2015; Post-Ebola – March 2017\n\n3 The overall recommended numbers of staff as per the BPEHS increased from 569 during the pre- and intra-Ebola period to 574 in the post-Ebola period as one new facility was added in the post-Ebola period.\n\n4 Similarly, during the post-Ebola period, 5 MCHPs were upgraded to CHPs increasing the staffing requirement for the CHPs from 288 to 318.\n\n\nDiscussion\n\nThis is the first study assessing staffing levels (medical and non-medical) 16 months into the post-Ebola period and comparing the status with pre- and intra-Ebola periods. The situation remains alarming with a 60% deficit for medical and 92% deficit for non-medical staff.\n\nWe need to reiterate our earlier urgent call for bold policies and donor support that goes beyond “business as usual.”3 In addition to enhancing staff training, further action could include rapid mobilization of financial resources for employment of non-medical and support staff, including those currently out of public service and reinstatement of retired medical personnel still fit enough to work2. Importantly the macro-economic restrictions on the wage bill imposed by the International Monetary Fund (IMF) hamper recruitment and adequate salary levels4. These need to be boldly tackled. Whether or not the BPEHS standards are realistic and adaptation thereof may also need consideration.\n\nThe strengths of the study are that we included all district public health facilities, all human resource cadres and similar data prior to, during and after the outbreak. The main limitation is that we might have excluded some staff not on regular payrolls (those working on a volunteer basis), although we believe this is unlikely to offset or negate our study findings.\n\nThere are two key messages from this study. First, at the current rate of 5% improvement in the medical staff deficit over the 16-month post-Ebola period (65% intra-Ebola to 60% post-Ebola), it will take an additional 12 years to achieve BPEHS standards - too little, too slow!\n\nSecond, the persistent 92% gap for non-medical staff has major implications for future Ebola and infectious disease outbreaks5. Essential services for infection prevention and control at health facilities and the implementation of personal hygiene measures and effective waste management depend on non-medical staff. In the unfortunate event of a new Ebola outbreak, the current scenario would result in a déjà vu of high transmission among health workers and the community at large6. Ending the restrictive wage bill4 is vital to mobilize the needed financial resources and rapidly employ and deploy staff.\n\nIn conclusion, with an overall health worker deficit of 1,018, 16 months into the post-Ebola period compared to a deficit of 1,026 during the Ebola outbreak, “nothing has really changed.” We reiterate our call for strong political will, international collaboration, generous funding and a change in hiring restrictions imposed by the IMF.\n\n\nData availability\n\nOpen Science Framework: Squire J. Squire_James_SORTIT2_HRH_data 2019. https://doi.org/10.17605/OSF.IO/QK5YG7.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\nThe Sierra Leone Health Management Information Systems, the District Health Information System 2 (DHIS2), is accessible with a Ministry of Health and Sanitation login through https://sl.dhis2.org/. The Directorate of Policy, Planning, and Information (DPPI) can be contacted through Dr. Francis Smart (drfsmart@gmail.com), Director, DPPI, MOHS, with an information request detailing the specific data request and purpose of use. Applicants will be asked to provide details of the reason for the request and details pertaining data request (such as data points, disaggregation, time period). In this case, data access would be granted to persons who request data for research purposes if they can provide appropriate ethical approval documentation.", "appendix": "Grant information\n\nThe programme was funded by the Special Programme for Research and Training in Tropical Diseases (TDR) hosted at the World Health Organization.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript...\n\n\nAcknowledgements\n\nThis research was conducted through the Structured Operational Research and Training Initiative (SORT IT), a global partnership coordinated by the Special Programme for Research and Training in Tropical Diseases at the World Health Organization (WHO/TDR) and implemented with partners. The training model is based on a course developed jointly by the International Union Against Tuberculosis and Lung Disease (The Union) and Medécins sans Frontières (MSF). The specific SORT IT programme which resulted in this publication was jointly developed and implemented by: WHO/TDR, the Sierra Leone Ministry of Health and Sanitation, WHO Sierra Leone and the Centre for Operational Research, The Union, Paris, France. Mentorship and the coordination/facilitation of the SORT IT workshops were provided through the Centre for Operational Research, The Union, Paris, France; Alliance for Public Health, Ukraine; Institute of Tropical Medicine, Antwerp, Belgium; and Sustainable Health Systems, Freetown, Sierra Leone.\n\n\nReferences\n\nMoHS: The Basic Package of Essential Health Services, 2015-2020. Freetown, Sierra Leone: Ministry of Health and Sanitation. 2015. Accessed 14th January, 2016. Reference Source\n\nSylvester Squire J, Hann K, Denisiuk O, et al.: The Ebola outbreak and staffing in public health facilities in rural Sierra Leone: who is left to do the job? Public Health Action. 2017; 7(Suppl 1): S47–S54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPhilips M, Markham A: Ebola: a failure of international collective action. Lancet. 2014; 384(9949): 1181. PubMed Abstract \| Publisher Full Text\n\nMarphatia A, Moussié R, Ainger A, et al.: Confronting the contradictions: The IMF, wage bill caps and the case for teachers. Action AID2007. Accessed 22 February 2019. Reference Source\n\nHarries AD, Zachariah R, Tayler-Smith K, et al.: Keeping health facilities safe: one way of strengthening the interaction between disease-specific programmes and health systems. Trop Med Int Health. 2010; 15(12): 1407–12. PubMed Abstract \| Publisher Full Text\n\nDallatomasina S, Crestani R, Sylvester Squire J, et al.: Ebola outbreak in rural West Africa: epidemiology, clinical features and outcomes. Trop Med Int Health. 2015; 20(4): 448–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSquire J: Squire_James_SORTIT2_HRH_data. osf.io/qk5yg. 2019. http://www.doi.org/10.17605/OSF.IO/QK5YG" }	[ { "id": "53417", "date": "04 Sep 2019", "name": "Wendemagegn Enbiale", "expertise": [ "Reviewer Expertise Human resource for health and Skin NTD" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have found the study very interesting and partially relevant in flagging the deficit for minimal staffing by label of care (based on the BPEHS standards) in Sierra Leone.\n\nSome of my question and concerns are:\nHealth Workforce (HWF) staffing usually measured based on population and there are intentional standards for developing countries. Why the authors are not interested to use or at least mention that?\n\nWhat is the catchment population of those health facilities and what is the health workforce staffing deficit based on the WHO criteria (for a developing country)?\n\nDo you need the HWF just to fulfil the BPEHS standard or is there any evidence suggesting compromisation of the routine health care service because of the stated HWF deficit.\n\nFor prioritization and focused action, I would recommend the showing the dis-aggregated deficit from nurses, midwifes and doctors which would be much more important for the policy maker for action.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "5071", "date": "09 Jan 2020", "name": "Katrina Hann", "role": "Author Response", "response": "Dear Dr Wendemagegn Enbiale, Thank you for your review and insightful comments on the manuscript. We have responded to each of your points below. 1. Reviewer comment 1. Health Workforce (HWF) staffing usually measured based on population and there are international standards for developing countries. Why the authors are not interested to use or at least mention that? Response: Thank you for this comment. The Basic Package for Essential Health Services (BPEHS) is the national standard used to guide health workforce requirements in Sierra Leone. Despite the existence of global methods usually using a population-based measure the BPEHS has been adapted to the Sierra Leone content and thus more appropriate to measure health workforce gaps. In particular, the BPEHS allows us to quantify the real gap between the Government’s commitment and the actual status in terms of staffing. In addition, the international standard for staffing measures focus only on clinical staff. However, in the Ebola and post-Ebola context, non-clinical staffing are equally essential, especially in terms of infection prevention and control measures. Therefore, the use of the BPEHS is more appropriate as it allows assessment of both medical and non-medical staff cadres. In the introduction, we have added more explanation and references to clarify our stand to use the BPEHS as the yardstick in the introduction. We have also added more information of the BPEHS standard in the methods section. 2. Reviewer Comment 2: What is the catchment population of those health facilities and what is the health workforce staffing deficit based on the WHO criteria (for a developing country)? Response: This study is focused on a specific district, Kailahun and all health facilities in the district was included. Details of the Study setting and population was already reported in the previous paper for which this study is a follow up. We have referenced this paper and its contents in case the reader might wish for more information. As justified in point 1 above, since we utilised the BPEHS national standards, we do not see applying the WHO criteria as relevant to this study. 3. Reviewer comment 3: Do you need the HWF just to fulfil the BPEHS standard or is there any evidence suggesting compromiation of the routine health care service because of the stated HWF deficit. Response: Ensuring basic standards of Human resources for health, is one of the WHO health systems building blocks. It is essential to achieve this requirement if health facilities are to have the “hands on deck” to ensure proper and effective functioning of health systems to support the delivery of care to the population. The 2014-2016 Ebola outbreak with the resulting decline in the health workforce resulted in the collapse of even the most basic of routine health services. The BPEHS is the national standard that stipulate the minimum staffing for each type of public health facility, which is critical to maintain service delivery standards and our study shows that we are far from these standards. This gap will need to be filled. We have now added references to support the evidence on the health workforce deficit and its detrimental effect on health service delivery. 4. Reviewer comment 4: For prioritization and focused action, I would recommend the showing the dis-aggregated deficit from nurses, midwifes and doctors which would be much more important for the policy maker for action. Response: Thank you for this important comment. In the already published article “The Ebola outbreak and staffing in public health facilities in rural Sierra Leone: who is left to do the job?” which has been referenced in the current study, we disaggregated the staffing deficit by cadre. Since there were no significant changes in the staffing levels, we decided not to duplicate the same results in the current study. We thank you for your kind understanding." } ] }, { "id": "53419", "date": "11 Sep 2019", "name": "Hayk Davtyan", "expertise": [ "Reviewer Expertise Operational research", "Tuberculosis", "HIV" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is very well written with clearly defined aim/research question and methodology designed to answer it. The question of interest is of high importance as it relates to Ebola (highly contagious and dangerous virus) which could become a threat for the whole world. The problem discussed in the article (under-staffing) is worrisome and calls for an urgent actions. It seems there is a need for more information on settings. Particularly regarding financing and hiring/staffing of the health facilities in the article. While the presentation of the problem is clear it will be really interesting and important to understand root-causes of the problems which may help to solve the issues discussed. Most importantly if there was a standard setting for minimum staff levels by the Ministry (so, assuming there is political will) then what are the constrains for not increasing number of human resources. The lack of qualified professionals may cause the under-staffing of medical resources but we observe no change in the number of non-medical staff so there should be other causes preventing the capacity increase in terms of human resources. If the data is not available researchers might want to address it in the limitations and call for further investigation of the topic.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5072", "date": "09 Jan 2020", "name": "Katrina Hann", "role": "Author Response", "response": "Dear Hayk Davtyan Many thanks for reviewing the manuscript and your insightful comments and contributions. We have provided responses to each of your comments below. The review comments are in bold font and we have responded using bullet points and normal font. Reviewer comment 1: It seems there is a need for more information on settings. Particularly regarding financing and hiring/staffing of the health facilities in the article. Response: Thank you for this important comment. As suggested, we have included more information on the settings specifically addressing your concerns. Reviewer comment 2. While the presentation of the problem is clear it will be really interesting and important to understand root-causes of the problems which may help to solve the issues discussed. Response: The current study’s aim and objectives were to identify what is the deficit (the gap) in the health workforce in relation to Basic stipulated standards and not to address root-causes of the deficit. In the discussion we mentioned the possible reasons and actions that needs to be taken to address some of the root causes. In future research, we hope to consider determining the root causes. Reviewer comment 3: Most importantly if there was a standard setting for minimum staff levels by the Ministry (so, assuming there is political will) then what are the constrains for not increasing number of human resources. Response: The BPEHS is the national standard that stipulates the minimum staffing for each type of public health facility, which is critical to maintain service delivery standards. The BPEHS target is to be achieved by 2020. However, with the current pace of implementation, this target will not be achieved by 2020. The constraints in increasing the health workforce are multiple and include limited available funds, availability of trained manpower, IMF restrictions etc. We have already highlighted the constraints and the urgent measures that need to be implemented to address the underlying staffing deficits. Reviewer comment 4: The lack of qualified professionals may cause the under-staffing of medical resources but we observe no change in the number of non-medical staff so there should be other causes preventing the capacity increase in terms of human resources. If the data is not available researchers might want to address it in the limitations and call for further investigation of the topic. Response: We have already discussed the underlying factors contributing to the observed deficits in both medical and non-medical staffing levels. We agree with the reviewer that more research is needed and have suggested areas that merit further research and investigation (in the Discussion)." } ] }, { "id": "53202", "date": "17 Sep 2019", "name": "Armand Sprecher", "expertise": [ "Reviewer Expertise Epidemiology", "filovirus outbreak management", "public health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study gives a simple, but clear, description of the gap between Sierra Leone's health structure staffing ambitions and the actual state of affairs in Kailahun over the last several years. By merely measuring the difference between what the ministry of health and sanitation has set as staffing objectives and what it has achieved, the study is no more complicated than it needs to be. As such, the methods and results sections are suitably short and to the point.\nThat being said, the paper would profit from going into greater depth in the discussion. This is hardly the first study to examine staffing shortfalls in Sierra Leone. The authors cite their own previous work, but there are other papers that merit mention in this domain. A quick PubMed search of \"Sierra Leone Staffing\" brings up a number of studies, some of which the authors may wish to include in their discussion of health structure staffing in Sierra Leone.\nThe reader is also left in the dark as to how the Basic Package of Essential Health Services was put together and whether the levels set in this standard were tailored to well assessed needs in Sierra Leone, imported from another context, or are aspirational. As such, it is difficult for the reader to guess at the impact of the measured gap.\nThe paper would benefit from some additional exploration of not meeting this standard. The authors speculate about the consequences of the severe non-medical under-staffing in terms of being unable to cope with infectious disease outbreaks because of insufficient hygiene staff, but at a 92% deficit of \"administrative staff, cleaners, cooks, maintenance workers, drivers, and security personnel\" (and this list should perhaps appear somewhere in the body of the paper and not just in a footnote to table 2), the day to day consequences should go well beyond this.\nThe reader might also profit from some information about why the gap has persisted. The recommendation to mobilize additional financial resources suggests that the underlying problem is insufficient funding, which seems reasonable, but the foundation for this recommendation is never laid by any mention of what has caused the gap to remain. Perhaps the money is available but the qualified people are in the middle of their training programs? Other problems could be at work as well, but the authors do not explore this next logical step following from their findings.\nThis is a nice simple study, but the readers would profit from the authors shedding more light on the meaning of their findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5073", "date": "09 Jan 2020", "name": "Katrina Hann", "role": "Author Response", "response": "Dear Armand Sprecher, Thanks for reviewing the manuscript and your insightful comments. We have provided responses to each of your comments below. Your comments are highlighted in bold and our responses follow using normal font. Reviewer comment 1. That being said, the paper would profit from going into greater depth in the discussion. This is hardly the first study to examine staffing shortfalls in Sierra Leone. The authors cite their own previous work, but there are other papers that merit mention in this domain. A quick PubMed search of \"Sierra Leone Staffing\" brings up a number of studies, some of which the authors may wish to include in their discussion of health structure staffing in Sierra Leone. Response: Thanks for the comment. We do agree that other studies have looked at staffing issues in Sierra Leone. However, this is the first study that has assessed the implementation of the BPEHS in relation to the Ebola outbreak and the references we have included in the paper relate to this theme. We thank you for your understanding. Reviewer comment 2: The reader is also left in the dark as to how the Basic Package of Essential Health Services was put together and whether he levels set in this standard were tailored to well assessed needs in Sierra Leone, imported from another context, or are aspirational. As such, it is difficult for the reader to guess at the impact of the measured gap. Response: The BPEHS is the national standard that stipulates the minimum staffing for each type of public health facility level, which is critical to maintain service delivery standards. The BPEHS has been described before and we have included its details and justification for its use in the introduction. The standards are not aspirational but a necessary requirement. Reviewer comment 3: The paper would benefit from some additional exploration of not meeting this standard. The authors speculate about the consequences of the severe non-medical under-staffing in terms of being unable to cope with infectious disease outbreaks because of insufficient hygiene staff, but at a 92% deficit of \"administrative staff, cleaners, cooks, maintenance workers, drivers, and security personnel\" (and this list should perhaps appear somewhere in the body of the paper and not just in a footnote to table 2), the day to day consequences should go well beyond this. Response. Thank you for this important comment. In the related article that was already published and duly referenced entitled “The Ebola outbreak and staffing in public health facilities in rural Sierra Leone: who is left to do the job?”, we disaggregated the staffing deficit by cadre. We cited this article in the current study. Since there were no significant changes in the staffing levels, we decided not to duplicate the same result in the current study. We have included a reference to the full list of medical and non-medical staff. Reviewer comment 4. The reader might also profit from some information about why the gap has persisted. The recommendation to mobilize additional financial resources suggests that the underlying problem is insufficient funding, which seems reasonable, but the foundation for this recommendation is never laid by any mention of what has caused the gap to remain. Perhaps the money is available but the qualified people are in the middle of their training programs? Other problems could be at work as well, but the authors do not explore this next logical step following from their findings. Response: In the discussion we have mentioned the possible reasons (which are multiple) and actions that needs to be taken to address some of the root causes and why corrective action has not been forthcoming. In future research, we hope to consider determining the root causes Reviewer comment 5: This is a nice simple study, but the readers would profit from e authors shedding more light on the meaning of their findings. Response: In the related article that was already published and duly referenced entitled “The Ebola outbreak and staffing in public health facilities in rural Sierra Leone: who is left to do the job?” we already touched the issues around IMF restrictions and retention issues and tried to avoid duplication in this current paper. We thank you for your kind consideration." } ] } ]	1	https://f1000research.com/articles/8-793
https://f1000research.com/articles/8-796/v1	06 Jun 19	{ "type": "Research Article", "title": "Paediatric morbidity and mortality in Sierra Leone. Have things changed after the 2014/2015 Ebola outbreak?", "authors": [ "Tom Sesay", "Olga Denisiuk", "Rony Zachariah", "Olga Denisiuk", "Rony Zachariah" ], "abstract": "Background: Sierra Leone was severely affected by the 2014/2015 Ebola outbreak and is likely to have had longer term repercussions on the health system including on paediatric morbidity and mortality. We thus assessed under-five morbidity and mortality for malaria, acute respiratory Infections (ARI)/pneumonia, watery diarrhoea and measles during the post-Ebola period in Sierra Leone and compared this with the pre- and intra-Ebola periods. Methods: This was a retrospective cross-sectional study using program data from the District Health Information system (DHIS2) and sourced from 14 districts in Sierra Leone. It included under-five children from 1,200 health facilities country-wide. Study periods included: before (June 1st, 2013-April 30th, 2014); during (June 1st, 2014-April 30th, 2015); and after Ebola (June 1st, 2016-April 30th, 2017). Results: Malaria, ARI/pneumonia and diarrhoea consultations declined during Ebola but recovered to pre-Ebola levels in the post-Ebola period. During the post-Ebola period, there was a highly significant reduction in case-fatality for the first three morbidities compared to the pre-Ebola period (P<0.0001). Average number of measles cases increased from 48/month in the pre-Ebola period to 568/month (12-fold increase) post-Ebola. Although there was no difference in measles case-fatality between the pre- and post-Ebola periods, case-fatality post-Ebola was significantly lower than during Ebola (Relative Risk: 0.05, 95% confidence interval 0.02-0.15, P<0.0001). Conclusions: Consultations for under-five children at health facilities in Sierra Leone recovered to pre-Ebola levels and case-fatality for common childhood illnesses declined significantly. This is a change for the better. However, the high level of reported measles cases in the post-Ebola period indicates gaps in immune status and needs focused attention.", "keywords": [ "Sustainable Development Goals", "Outbreak response", "Universal Health Coverage", "Operational Research", "SORT IT" ], "content": "Introduction\n\nIn 2017, a cross-sectional study1 documented country-wide morbidity for four common childhood illnesses: malaria, acute respiratory infections (ARI)/pneumonia, watery diarrhoea and measles. There were two main findings. First, during the Ebola outbreak, health facility visits for malaria, ARI and watery diarrhoea dropped significantly nation-wide, without returning to pre-Ebola levels post-outbreak. As these morbidities have similar symptom patterns as Ebola, people may have avoided accessing formal health services to avoid being considered “an Ebola case”. Second, measles cases increased dramatically by six-fold during Ebola and the immediate post-Ebola periods. This was attributed to cessation of measles vaccination activities during the Ebola outbreak.\n\nThe outbreak was declared over in November 2015. Since then, there have been considerable investments into the health system by Government and development partners. One of the limitations of the 2017 study1 was that it only included the immediate six-month period after the Ebola outbreak, which might have been too early to assess health system recovery. It is now expected that the country would have fully recovered from the outbreak, but there has been no formal evaluation in this regard.\n\nWe thus conducted a similar country-wide study assessing morbidity and mortality for the same childhood illnesses using a longer post-Ebola period and compared this data with the pre- and intra Ebola periods.\n\n\nMethods\n\nThis was a retrospective analysis using routine program data from the District Health Information system (DHIS2) and sourced from all 14 districts in Sierra Leone (see Underlying data2).\n\nThe study setting was described in detail before1. In brief, the health infrastructure is tiered into tertiary hospitals, district hospitals and Peripheral Health Units (PHUs). PHUs include Community Health Centres (CHCs), Community Health Posts (CHPs) and Maternal and Child Health Posts (MCHPs). The Ministry of Health and Sanitation provides free primary health care for children under five across 1,250 health facilities nationwide.\n\nThe study population included all children under-five years attending public health facilities nationwide. No children were excluded.\n\nStudy periods included: before (June 1st 2013-April 30th 2014); during (June 1st 2014-April 30th 2015); and after Ebola (June 1st 2016-April 30th 2017).\n\nWe exported data on health facility visits and mortality for malaria, (ARI)/pneumonia, watery diarrhoea and measles from the DHIS2 to Microsoft excel (2016) for analysis. Differences between groups were assessed using Pearson’s X2 test (Chi square). Levels of significance were set at P ≤ 0.05.\n\nEthics approval was obtained from the Sierra Leone Ethics and Scientific Review Board (dated 14 December 2018) and the Union Ethics Advisory Group (International Union against Tuberculosis and Lung Disease, Paris, France; EAG number: 68/18). As the study used anonymous data, there was no need for informed consent.\n\n\nResults\n\nFigure 1 shows country-wide trends in outpatient consultations for malaria, ARI, watery diarrhoea and measles. Consultations followed a seasonal pattern with an overall decline during Ebola. In the post-Ebola period (assessed six months after the end of the outbreak), consultations reached pre-Ebola levels. In contrast, measles increased during the last six months of the Ebola outbreak and this trend continued into the post-Ebola period. Average numbers of measles cases were 48/month in the pre-Ebola period, increasing to 87/month in the Ebola period and 568/month (12-fold increase) post-Ebola. Measles cases peaked in March 2017 with 853 cases.\n\nTable 1 shows numbers of cases, deaths and case-fatality (per 1000) for malaria, ARI/pneumonia, watery diarrhoea and measles. During the post-Ebola period, there was a highly significant reduction in case-fatality for the first three morbidities compared to the pre-Ebola period (P<0.0001).\n\n1 Pre-Ebola: June 1st 2013 – April 30th 2014; Ebola: June 1st 2014 – April 30th 2015; Post-Ebola: June 1st 2016 – April 30th 2017.\n\n2 Chi-square comparing the pre-Ebola and post-Ebola periods.\n\nFor measles, there was a total of 525 cases pre-Ebola, 962 cases during Ebola and 6245 cases post-Ebola. Although there was no difference in measles case-fatality between the pre- and post-Ebola periods case-fatality post-Ebola was significantly lower than during Ebola (Relative Risk: 0.05, 95% confidence interval 0.02–0.15, P<0.0001).\n\n\nDiscussion\n\nThis study shows that health facility consultations for malaria, ARI/Pneumonia and watery diarrhoea recovered to pre-Ebola levels and were accompanied by significant country-wide reductions in case-fatality compared to the pre-Ebola period. Despite a dramatic increase in measles cases post-Ebola, there was a significant mortality reduction, suggesting overall improvements in clinical care.\n\nA study strength is that we included data from 1250 health facilities and for similar periods before, during and after the outbreak. A limitation is that our data did not include private health facilities resulting in possible underestimation of disease burden.\n\nThere were two key findings. First, the reductions in case fatality from malaria, ARI and watery diarrhoea could be associated with improved post-Ebola health seeking behaviour and re-establishment of community confidence in the health system. The post-Ebola recovery plan3 of the Government of Sierra Leone with enhanced financial, technical and training support from partners may also have contributed. Furthermore, community health worker activities including early identification and referrals of ill children were promoted which in turn would contribute to reducing mortality.\n\nSecond, increased measles cases during and after Ebola could be attributed to vaccination service cessation during Ebola in line with the recommendation to avoid invasive procedures as a way of minimizing Ebola-related occupational risks4. Many children would have missed their measles vaccination, resulting in a reduction in herd immunity as well as an accumulation of unvaccinated children. Measles coverage among children under two years in 2017 (post-Ebola) stood at 80%5 while pre-Ebola this was at a low 78.6%6. This implies that measles vaccination coverage was already below the desired level prior to Ebola and remained below desired levels after Ebola. This calls for measles vaccination campaigns at a higher age cut-off (6 months–15 years) to increase vaccination coverage to at least 95%7,8. Laboratory confirmation is also needed to ensure that reported cases are not being mis-diagnosed since measles diagnosis is largely clinical.\n\nIn conclusion, consultations of under-five children at health facilities in Sierra Leone recovered to pre-Ebola levels and case-fatality for common childhood illnesses declined significantly. This is a change for the better. However, the high level of reported measles cases in the post-Ebola period needs focused attention.\n\n\nData availability\n\nThe Sierra Leone Health Management Information Systems, the District Health Information System 2 (DHIS2), is accessible with a Ministry of Health and Sanitation (MOHS) login through https://sl.dhis2.org/. The Directorate of Policy, Planning, and Information (DPPI) can be contacted to arrange access through Dr. Francis Smart (drfsmart@gmail.com), Director, DPPI, MOHS.\n\nRepository: Dataset 1. Sesay_Tom_SORTIT2_paed_data. https://doi.org/10.17605/OSF.IO/SYP7G2\n\nThis project contains the following underlying data:\n\nSesay_T_casefatality_data.xlsx (case fatality data)\n\nSesay_T_morbidity_data.xlsx (morbidity data)\n\nSesay_T_paed_datadictionary.xlsx (data dictionary)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThe programme was funded by the Special Programme for Research and Training in Tropical Diseases hosted at the World Health Organization (TDR).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgement\n\nThis research was conducted through the Structured Operational Research and Training Initiative (SORT IT), a global partnership coordinated by the Special Programme for Research and Training in Tropical Diseases at the World Health Organization (WHO/TDR) and implemented with partners. The training model is based on a course developed jointly by the International Union Against Tuberculosis and Lung Disease (The Union) and Medécins sans Frontières (MSF). The specific SORT IT programme which resulted in this publication was jointly developed and implemented by: WHO/TDR, the Sierra Leone Ministry of Health and Sanitation, WHO Sierra Leone and the Centre for Operational Research, The Union, Paris, France, Alliance for Public Health, Ukraine; Institute of Tropical Medicine, Antwerp, Belgium; and Sustainable Health Systems, Freetown, Sierra Leone.\n\n\nReferences\n\nSesay T, Denisiuk O, Shringarpure KK, et al.: Paediatric care in relation to the 2014-2015 Ebola outbreak and general reporting of deaths in Sierra Leone. Public Health Action. 2017; 7(Suppl 1): S34–S9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSesay T: Sesay_Tom_SORTIT2_paed_data. 2019. http://www.doi.org/10.17605/OSF.IO/SYP7G\n\nNational Ebola Recovery Strategy for Sierra Leone, 2015-2017. (Accessed 20th February 2019). Reference Source\n\nTakahashi S, Metcalf CJ, Ferrari MJ, et al.: Reduced vaccination and the risk of measles and other childhood infections post-Ebola. Science. 2015; 347(6227): 1240–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMICS: Sierra Leone multiple indictor cluster survey 2017, Survey findings report, Statistics Sierra Leone. (Accessed 20nd February, 2019). Reference Source\n\nStatistics Sierra Leone (SSL) and ICF International: Sierra Leone Demographic and Health Survey 2013. Freetown, Sierra Leone and Rockville, Maryland, USA: SSL and ICF International. 2014. Reference Source\n\nTruelove SA, Moss WJ, Lessler J: Mitigating measles outbreaks in West Africa post-Ebola. Expert Rev Anti Infect Ther. 2015; 13(11): 1299–301. PubMed Abstract \| Publisher Full Text\n\nLuquero FJ, Pham-Orsetti H, Cummings DA, et al.: A long-lasting measles epidemic in Maroua, Cameroon 2008-2009: mass vaccination as response to the epidemic. J Infect Dis. 2011; 204 Suppl 1: S243–51. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "52581", "date": "08 Oct 2019", "name": "Sara Hersey", "expertise": [ "Reviewer Expertise Infectious disease epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors of this paper have looked at trends in the pediatric morbidity and mortality prior to, during and after the 2014-15 Ebola outbreak in West Africa. The impact of large scale outbreaks on health systems remains a critical area of research, and the authors should be congratulated in contributing to this area of work. The comments below are intended to better contextualize the analysis and conclusions proposed by the authors.\nThe authors divide the paper into three distinct time periods, with ‘post-Ebola’ defined as beginning in June 2016 and extending through April 2017. While the end of the Ebola outbreak in Sierra Leone was declared in November 2015, there continued to be additional Ebola clusters in the country and the region into the ‘post-Ebola’ study period. The policy of mandatory Ebola testing for all deaths also continued until June 2016.\n\nDuring the ‘post-Ebola’ time period in the paper, there was an enormous influx of funding to improve health systems at both the facility and community levels that was organized under the Presidential Recovery Plan. This funding supported the national and sub-national health systems but was also supplemented by NGO and other non-governmental health services. By the end of 2017, there were substantial reductions in the funds available for health systems and service delivery.\n\nThe continued specter of Ebola transmission and the rapid scale-up in health resources likely had both negative and positive impacts on health systems utilization that are difficult to quantify. The authors have documented significantly lower case fatality rates for three diseases than the pre-Ebola time period which would support the conclusion that there was regained community trust in the health services. However, those services were not the same ones available prior to the Ebola outbreak and the services are difficult to maintain with reduced external resources.\n\nIn order to compare the pre and post-Ebola periods, I would recommend that the analysis be extended to the immediate post-Ebola period included in this paper as well as a longer-term post-Ebola period through 2018 which would better capture the more sustainable health system in Sierra Leone rather than the one served by a short-term, high-volume influx of external funding.\n\nAside from the clear spikes in measles consultations, Figure 1 is difficult to interpret when displayed by time period. The differences may be more impactful if compared by disease instead.\n\nThe analysis supports the conclusion that measles surged during and post-Ebola and this is backed up by other research and program documentation. However, it does not necessarily support the recommendation to extend the cut off age for vaccination. The recommendation to conduct laboratory confirmation for measles is also not necessarily supported by the data presented. Is this because a number of the measles outbreaks were determined to be rubella which is currently not on the vaccine schedule? If yes, this should be documented by the data. What would be the impact on measles morbidity if there was increased diagnostic versus syndromic management of measles?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5086", "date": "09 Jan 2020", "name": "Tom Sesay", "role": "Author Response", "response": "Author Response to Reviewer Report for Version 1 from Sara Hersey 08 Oct 2019 Thank you for your thoughtful review of the manuscript and your insightful suggestions. We have responded as below. 1. Reviewer comment:The authors of this paper have looked at trends in the pediatric morbidity and mortality prior to, during and after the 2014-15 Ebola outbreak in West Africa. The impact of large scale outbreaks on health systems remains a critical area of research, and the authors should be congratulated in contributing to this area of work. The comments below are intended to better contextualize the analysis and conclusions proposed by the authors.Response:Thank you for recognising the importance of this study and its contribution to knowledge about the impact of the 2014/15 Ebola outbreak. 2. Reviewer comment:The authors divide the paper into three distinct time periods, with ‘post-Ebola’ defined as beginning in June 2016 and extending through April 2017. While the end of the Ebola outbreak in Sierra Leone was declared in November 2015, there continued to be additional Ebola clusters in the country and the region into the ‘post-Ebola’ study period. The policy of mandatory Ebola testing for all deaths also continued until June 2016. During the ‘post-Ebola’ time period in the paper, there was an enormous influx of funding to improve health systems at both the facility and community levels that was organized under the Presidential Recovery Plan. This funding supported the national and sub-national health systems but was also supplemented by NGO and other non-governmental health services. By the end of 2017, there were substantial reductions in the funds available for health systems and service delivery. The continued specter of Ebola transmission and the rapid scale-up in health resources likely had both negative and positive impacts on health systems utilization that are difficult to quantify. The authors have documented significantly lower case fatality rates for three diseases than the pre-Ebola time period which would support the conclusion that there was regained community trust in the health services. However, those services were not the same ones available prior to the Ebola outbreak and the services are difficult to maintain with reduced external resources. In order to compare the pre and post-Ebola periods, I would recommend that the analysis be extended to the immediate post-Ebola period included in this paper as well as a longer-term post-Ebola period through 2018 which would better capture the more sustainable health system in Sierra Leone rather than the one served by a short-term, high-volume influx of external funding.Response:Thank you for this recommendation which we do understand and accept. We do acknowledge the need for further research into the sustainability of the early post-Ebola gains in a much longer post-Ebola period. However, the objective of this study was to assess whether the investments in the health system had an impact on service delivery in the post-Ebola period, as defined in the manuscript. In addition, the ethical approval obtained for this study was only for the periods mentioned in the paper. This, however, is an area we will be interested to investigate in the near future. We have added a recommendation to this effect in the discussion section. 3. Reviewer comment:Aside from the clear spikes in measles consultations, Figure 1 is difficult to interpret when displayed by time period. The differences may be more impactful if compared by disease instead.Response: We agree with your recommendation, and accordingly have completed the analysis separately by the disease conditions. We have amended the Figure 1 based on this. 4. Reviewer comment:The analysis supports the conclusion that measles surged during and post-Ebola and this is backed up by other research and program documentation. However, it does not necessarily support the recommendation to extend the cut off age for vaccination. The recommendation to conduct laboratory confirmation for measles is also not necessarily supported by the data presented. Is this because a number of the measles outbreaks were determined to be rubella which is currently not on the vaccine schedule? If yes, this should be documented by the data. What would be the impact on measles morbidity if there was increased diagnostic versus syndromic management of measles?Response:We do agree with this recommendation. We have removed the recommendation to extend the cut off age for vaccination and added the recommendation to increase the coverage of the second measles dose at 15 months." } ] }, { "id": "52585", "date": "28 Oct 2019", "name": "Justin Lessler", "expertise": [ "Reviewer Expertise Epidemiology", "statistics", "infectious disease dynamics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n\"Paediatric morbidity and mortality in Sierra Leone. Have things changed after the 2014/2015 Ebola outbreak?\" provides a straight forward analysis of changes in reports of, and mortality from, four diseases before, during and after the 2014/2015 Ebola outbreak based on data from DHIS2 in Sierra Leone. The report provides simple, but important, statistics on morbidity and mortality for malaria, ARI/pneumonia, watery diarrhea, and measles. While the data and analysis is important, I have some significant concerns with the statistical analysis and interpretation of the results:\nStatistics: While the use of chi-squared test to identify that significant differences in mortality exist is appropriate, it only is being used to compare pre- and post Ebola periods and does not help with evaluation of the size of the changes. I would suggest the authors instead select a reference period and report relative rates and confidence intervals, so the data can be better understood. Further, the authors do not perform statistical tests to support some of their statements, some of which are noted below. More tests for differences in reported rates between periods and some analysis of changes in the Ebola period would better support their conclusions.\nInterpretation: The authors make several statements in the discussion and abstract that do not correspond to the analyses done. For instance, they make statements regarding trends in case reports, but perform no statistical test as to whether between period changes in reports are significant. Further, in some cases they frame speculation as a key finding of the paper, such as when they attribute changes to community confidence in the health system. Such speculations need to be clearly framed as such.\nOverall, this paper could be made far stronger and more valuable with the addition of just a few additional basic statistical analyses that were more aligned with the conclusions the authors which to highlight in the discussion. This analysis could likely be done while preserving the admirable conciseness and brevity of the paper.\n\nSpecific notes:\n\nIntroduction, \"to early to assess...\": Could this not just be recovery, but also improvements, as significant investments followed the Ebola outbreak.\n\nDiscussion, \"First, the reductions in case fatality...\": There is not really any evidence for this association in the paper, it should be, at the very least, more clear this is speculation.\n\nDiscussion, \"Second, increased measles cases...\": If this is going to be said it needs to be better supported by the statistical analysis in the paper.\n\nDiscussion, \"In conclusion, consultations...\": This was not explicitly tested in the analysis, so I am not sure it can be concluded.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "5087", "date": "09 Jan 2020", "name": "Tom Sesay", "role": "Author Response", "response": "Author Response to Reviewer Report for Version 1 from Justin Lessler 28 Oct 2019 Thank you for your thoughtful review of the manuscript and your suggestions. We have responded as below. 1. Reviewer comment:\"Paediatric morbidity and mortality in Sierra Leone. Have things changed after the 2014/2015 Ebola outbreak?\" provides a straight forward analysis of changes in reports of, and mortality from, four diseases before, during and after the 2014/2015 Ebola outbreak based on data from DHIS2 in Sierra Leone. The report provides simple, but important, statistics on morbidity and mortality for malaria, ARI/pneumonia, watery diarrhea, and measles.Response:Thank you for this comment. 2. Reviewer comment: While the data and analysis is important, I have some significant concerns with the statistical analysis and interpretation of the results. Statistics: While the use of chi-squared test to identify that significant differences in mortality exist is appropriate, it only is being used to compare pre- and post Ebola periods and does not help with evaluation of the size of the changes. I would suggest the authors instead select a reference period and report relative rates and confidence intervals, so the data can be better understood. Further, the authors do not perform statistical tests to support some of their statements, some of which are noted below. More tests for differences in reported rates between periods and some analysis of changes in the Ebola period would better support their conclusions.Response: Thank you for this point. We have performed t-tests for the differences in the mean monthly cases of the intra-Ebola and post-Ebola periods relative to the pre-Ebola period at a level of significance of 5%. We have also updated the figures by disease condition, which better displays the differences between the periods. 3. Reviewer comment:Interpretation: The authors make several statements in the discussion and abstract that do not correspond to the analyses done. For instance, they make statements regarding trends in case reports, but perform no statistical test as to whether between period changes in reports are significant. Further, in some cases they frame speculation as a key finding of the paper, such as when they attribute changes to community confidence in the health system. Such speculations need to be clearly framed as such.Response: We have taken note of this comment and, accordingly, removed comments that could be considered as speculations. 4. Reviewer comment:Overall, this paper could be made far stronger and more valuable with the addition of just a few additional basic statistical analyses that were more aligned with the conclusions the authors which to highlight in the discussion. This analysis could likely be done while preserving the admirable conciseness and brevity of the paper. Response: We have included the basic statistical tests to test the statistical significance of differences between periods, as mentioned above. 5. Reviewer comment:Introduction, \"to early to assess...\": Could this not just be recovery, but also improvements, as significant investments followed the Ebola outbreak.Response: This comment is noted, and we have added in a phrase on possible improvements. 6. Reviewer comment:Discussion, \"First, the reductions in case fatality...” There is not really any evidence for this association in the paper, it should be, at the very least, more clear this is speculation. Response: The reductions in case fatality were indicated by a Chi-square test of the difference between the pre-Ebola and the post-Ebola period which showed a significant reduction in case fatality in the post- Ebola period. 7. Reviewer comment:Discussion, \"Second, increased measles cases...\": If this is going to be said it needs to be better supported by the statistical analysis in the paper. Response: We have included a t-test of the average monthly measles cases, which showed significant increase in the number of measles cases between the pre-Ebola and post-Ebola periods. 8. Reviewer comment:Discussion, \"In conclusion, consultations...\": This was not explicitly tested in the analysis, so I am not sure it can be concluded.Response: We have ensured that the relevant statistical tests have been completed, with corresponding results to support the conclusions." } ] } ]	1	https://f1000research.com/articles/8-796
https://f1000research.com/articles/8-1586/v1	04 Sep 19	{ "type": "Review", "title": "Biological effects of nicotine exposure: A narrative review of the scientific literature", "authors": [ "Leonie R. Price", "Javier Martinez", "Javier Martinez" ], "abstract": "The emergence of new tobacco heating products and electronic nicotine delivery systems (ENDS) is changing the way humans are exposed to nicotine. The purpose of this narrative review is to provide a broad overview of published scientific literature with respect to the effects of nicotine on three key health-related areas: 1) cardiovascular risk, 2) carcinogenesis and 3) reproductive outcomes. These areas are known to be particularly vulnerable to the effects of cigarette smoke, and in addition, nicotine has been hypothesized to play a role in disease pathogenesis. Acute toxicity will also be discussed.\n\nThe literature to February 2019 suggests that there is no increased cardiovascular risk of nicotine exposure in consumers who have no underlying cardiovascular pathology. There is scientific consensus that nicotine is not a direct or complete carcinogen, however, it remains to be established whether it plays some role in human cancer propagation and metastasis. These cancer progression pathways have been proposed in models in vitro and in transgenic rodent lines in vivo but have not been demonstrated in cases of human cancer.\n\nFurther studies are needed to determine whether nicotine is linked to decreased fertility in humans. The results from animal studies indicate that nicotine has the potential to act across many mechanisms during fetal development. More studies are needed to address questions regarding nicotine exposure in humans, and this may lead to additional guidance concerning new ENDS entering the market.", "keywords": [ "Nicotine", "Electronic Nicotine Delivery Systems", "Cardiovascular Diseases", "Carcinogenesis", "Fetal Development", "Fertility", "Acute Toxicity", "Nicotine Replacement Therapy" ], "content": "Introduction\n\nNewly developed tobacco and nicotine-containing products such as e-cigarettes are being widely accepted by consumers. In order to analyze any potential pathological roles of nicotine, there is a need to consider it in both isolation and within the new landscape of emerging tobacco heating products and electronic nicotine delivery systems (ENDS). It is already understood that nicotine is not a major contributing factor to the diseases associated with tobacco smoking (Benowitz et al., 1998; Gottlieb & Zeller, 2017; Royal College of Physicians, 2016) and although much investigation has already been carried out, nicotine research continues to be an active field of study. This review will provide an overview of the research up to February 2019, giving insight into the roles of nicotine in the development of acute toxicity, and discuss whether nicotine may adversely affect health in three specific areas: carcinogenesis, cardiovascular risk and reproduction. These areas are known to be particularly vulnerable to the effects of cigarette smoke, and in addition, nicotine has been hypothesized to play a role in disease pathogenesis.\n\nExtensive analysis into nicotine as a pharmacological molecule has already been carried out (Baraona et al., 2017; Benowitz, 1996; Mishra et al., 2015; West, 2017). This review will attempt not to duplicate existing publications, and instead will focus on the potential effects of nicotine within the context of emerging products and nicotine replacement therapies.\n\n\nSearch criteria\n\nPubMed was used to carry out literature searches from 2009 to February 2019 with the following search terms: nicotine carcinogenesis, nicotine replacement therapies cancer, nicotine cardiovascular, nicotine acute toxicity cardiovascular, nicotine acute toxicity, chronic exposure to nicotine, nicotine thrombosis, nicotine pregnancy outcomes, nicotine pregnancy, and nicotine fertility.\n\nStudies citing tobacco smoking nicotine exposure were either excluded or addressed cautiously as appropriate. The most recent studies and those carried out in human participants were prioritized where possible, although references to animal and cell studies were necessary, given some lack of studies in humans. Relevant studies and studies prior to 2009 referenced in the returned papers were included. Additional relevant studies from JTI databases were also added. It should be noted that this study was not designed as a systematic review. For full search results, methodology, exclusion criteria, and flow diagram, please see supplementary methods (Extended data (Price & Martinez, 2019)).\n\n\nAcute nicotine toxicity\n\nThe symptoms of nicotine poisoning, or toxicity are well known and have been characterized through case studies (Franke & Thomas, 1936; Paik et al., 2018), as well as the study of green tobacco sickness (GTS) (Gehlbach et al., 1974). GTS is the manifestation of acute nicotine toxicity typically identified in tobacco workers who have transdermally absorbed high levels of nicotine from handling tobacco leaves. The symptoms include nausea, vomiting, headache, abdominal cramps, breathing difficulty, abnormal body temperature, pallor, diarrhea, chills, fluctuations in blood pressure and heart rate, sweats and increased salivation. It has been demonstrated that the concentration of salivary cotinine, a primary metabolite of nicotine, correlates with the four main symptoms of GTS (headache, nausea, vomiting and dizziness) (Saleeon et al., 2016). The acute symptoms of GTS pass as nicotine is metabolized, however, the long-term effects of GTS are unknown. Within the tobacco-handling work force, multiple GTS incidences may occur in the same individual, and therefore, further research is essential to understand whether there may be any long-term health impact as a result of acute nicotine toxicity.\n\nCurrent safety databases state that the lethal dose of ingested nicotine in adults is 60 mg (Cameron et al., 2014), which is equivalent to the amount found in approximately five cigarettes (Mayer, 2014). Despite the low lethal dose and high availability of nicotine in various forms, there are relatively few cases of fatal overdose reported. Conversely, there are many reports of survival after significantly higher exposures (Solarino et al., 2010). After a careful examination of historical reports by Mayer, it seems that the 60 mg dose was derived from self-experimentation carried out in 1857 by Reil, which does not appear to be highly reliable, but is still highly relied upon (Mayer, 2014). In the intervening period, the 60 mg threshold has become deeply rooted, and therefore, neither questioned nor updated.\n\nSymptoms of acute toxicity have been demonstrated in studies assessing the effects of nicotine replacement therapies (NRTs). In a study investigating the cardiovascular effects of transdermal nicotine patches (discussed in more detail later), participants were administered nicotine using up to three patches equating to 63 mg nicotine (interestingly higher than the ‘lethal’ dose, although not ingested). The authors had to change their study protocol because in the highest dose group, nausea and vomiting were experienced 2-4 hours after patch application, corresponding to peak plasma nicotine concentration. For this reason, patches had to be applied at staggered intervals to avoid the toxic effects (Zevin et al., 1998).\n\nNon-smokers often report mild symptoms of nicotine toxicity after exposure to very low levels (2–5 mg), but resistance to the symptoms develops rapidly on repeat exposure and varies extensively between individuals (Solarino et al., 2010).\n\nSince ENDS were introduced to the market, the number of reported incidences of nicotine poisoning have increased in both Europe (Vardavas et al., 2017) and the USA (Chatham-Stephens et al., 2016; Mowry et al., 2015). The route of exposure for the majority of these cases were ingestion, inhalation, ocular or dermal, which occurred predominantly in children under the age of five (Chatham-Stephens et al., 2016). Interestingly, Vardavas et al. note that “The number of incidents reported to national poison centers were not proportional to either the country’s population or the prevalence of e-cigarette use”. Overall, these reports indicate that acute toxicity from nicotine is highly unlikely to occur when ENDS are used as intended by adults.\n\n\nCardiovascular effects\n\nNicotine primarily acts on the cardiovascular system through stimulation of the sympathetic nervous system leading to release of norepinephrine and increases in heart rate, blood pressure, myocardial contractility and systemic vasoconstriction (Figure 1) (Benowitz & Burbank, 2016; Haass & Kübler, 1997). Many studies have investigated the relationship between smoking and increased cardiovascular risk, but few investigate nicotine and cardiovascular risk in humans.\n\n1) Nicotine binds to the nicotinic acetylcholine receptor (nAChR) causing a conformational change. 2) Sodium ions (Na+) enter the cell through the central pore of the nAChR. 3) Ion exchange leads to influx of calcium ions (Ca2+) into the cell causing depolarization. 4) Depolarization stimulates exocytosis of stored norepinephrine. 5) Norepinephrine binds to α1 adrenergic receptors on the smooth muscle cell surface, stimulating calcium ion influx and release of stored calcium. 6) Norepinephrine also binds to α2 adrenergic receptors on the smooth muscle cell surface, which causes downstream inhibition of adenylyl cyclase activity. Both these mechanisms cause smooth muscle contraction and, consequently, vasoconstriction.\n\nVansickel et al. carried out a study in 20 men and women of various racial backgrounds. All the participants smoked and were healthy. Five minutes after six puffing bouts of an e-cigarette, plasma nicotine increased from a baseline of 2.2 ng/ml to 7.4 ng/ml. Plasma nicotine was only significantly increased after four puffing bouts; heart rate was elevated after the first and second but returned to baseline by the third puffing bout. There was no effect on either systolic or diastolic blood pressure (Vansickel et al., 2012). This study indicated that there is little cardiovascular effect experienced after use of an e-cigarette, although it should be noted that the plasma nicotine levels achieved in this study were lower than those observed in cigarette smokers, which typically peak at 15–25 ng/ml. Nicotine is not the only ingredient in e-liquids. It has been reported that other ingredients, such as propylene glycol, may also have an effect on the cardiovascular system (Chaumont et al., 2018); however, an important recent study by Moheimani et al. examined the acute sympathetic roles of the nicotine and non-nicotine based constituents in e-liquids. Despite difficulties inducing an increase in plasma nicotine levels in their participants, the authors observed an increase in sympathetic activity and an increase in heart rate in those who smoked an e-cigarette with nicotine versus without or a sham puff. There was no significant difference in systolic, diastolic or mean arterial blood pressures or measures of oxidative stress and inflammation (Moheimani et al., 2017a). The authors conclude that unlike conventional cigarettes, nicotine is the sole ingredient in e-cigarettes responsible for inducing cardiovascular response. Results published by Franzen et al. support this finding. The authors showed that vaping an e-cigarette containing 24 ng/ml nicotine led to significant transient increases in systolic blood pressure, diastolic blood pressure and heart rate. In contrast, vaping a nicotine-free e-cigarette led to no change in systolic blood pressure, and transient small decreases in diastolic blood pressure and heart rate (Franzen et al., 2018). In another study, Moheimani et al. also found that long-term e-cigarette users showed decreased heart-rate variability and increased oxidative stress compared to non-users, both of which are markers for cardiovascular risk. The authors eliminated the possibility of acute nicotine interference by instructing their participants to abstain from using their e-cigarettes for 12 hours prior to testing. They acknowledged that most of their e-cigarette users were former smokers, and this could have had a chronic effect on heart-rate variability, although they state that this was unlikely to be responsible for the observations from the study (Moheimani et al., 2017b).\n\nFarsalinos et al. showed that smokers with high blood pressure who reduced their smoking by switching to e-cigarettes showed a significant reduction in systolic blood pressure after 1 year. This suggests that the nicotine found in both cigarettes and e-cigarettes is not responsible (at least entirely) for the chronic increased blood pressure found in smokers. There are too many variables in this study to isolate the effects of nicotine alone, including the fact that very few of the participants stopped smoking conventional cigarettes completely (Farsalinos et al., 2016).\n\nA key study investigated cardiovascular parameters, such as blood pressure, in smokers who switched from conventional cigarettes to e-cigarettes exclusively for five days. The authors estimated the nicotine exposures from the e-cigarette group to be similar to those in the conventional cigarette group, based on usage. Nevertheless, a significant reduction in heart rate, and systolic blood pressure was observed in the e-cigarette group (D’Ruiz et al., 2017). The results indicate that nicotine was not responsible for the cardiovascular profile of the participants, as changes were observed despite nicotine exposure remaining similar across all study groups. The authors concluded that other constituents in conventional cigarettes are more likely to cause the damage to cardiovascular health observed in smokers (D’Ruiz et al., 2017). Conversely, when non-smokers smoked a single cigarette and then an e-cigarette one week later, similar vascular effects were observed in response to both products. There was a significant decrease in flow-mediated dilation and an increase in markers of endothelial dysfunction after both the cigarette and e-cigarette, although the changes after e-cigarette use were smaller (Carnevale et al., 2016). The authors suggest that nicotine could be the cause of these changes, although the effects of other e-liquid components (such as flavorings, propylene glycol and glycerin), could not be ruled out. Chaumont et al. also raised this point in their paper, where they stated, “The pharmacological actions of nicotine make it impossible to distinguish the respective effects of the carriers themselves (propylene glycol and glycerin) and nicotine on the endothelial dysfunction, oxidative stress and arterial stiffness increase they observed”. To address this, the group carried out a study to assess vascular parameters in response to vaping with nicotine, vaping without nicotine and sham vaping in a small group of 25 non-daily smokers. They observed a significant decrease in acetylcholine-mediated vasodilation, as well as an increase in heart rate, diastolic and systolic blood pressure after nicotine vaping vs. baseline, for up to 120 minutes after use. An effect was also seen after use of the non-nicotine-containing e-liquid, but this was only significant for heart rate during vaping. It is not clear whether there was a significant difference between nicotine and non-nicotine-containing e-liquids as this statistical comparison was not carried out; the authors compared only to baseline. The vaping protocol used in this study was intense and used high-powered e-cigarette devices. The authors did not measure plasma nicotine concentrations in their participants, but the consumption of e-liquid far exceeded that of “regular vapers’ habits”. Once again, this study looked only at the acute effects (Chaumont et al., 2018); similar acute responses to nicotine-containing e-cigarettes have also been shown in additional studies (Franzen et al., 2018; Ikonomidis et al., 2018; Joesbury et al., 1998; Vlachopoulos et al., 2016; Yan & D’Ruiz, 2015), as well as with NRT (Johnston et al., 2018; Sabha et al., 2000).\n\nIn the first study of its kind, Polosa et al. followed a small group of e-cigarette users who had never smoked (n = 9; six of whom used nicotine-containing e-liquid), over a period of 3.5 years. At baseline, there were no differences between the vapers and their age/sex-matched controls for all parameters except heart rate (vapers had a slightly lower heart rate compared to the controls). After 3.5 years, there were no changes to any of the cardiovascular parameters measured (heart rate, systolic or diastolic blood pressure) in any of the participants, including those who used the nicotine-containing e-liquid. The participants were all young and healthy (average age 26.6 and 27.8 years for the EC users and controls respectively), and therefore were not at high risk of cardiovascular outcomes. The number of participants was also very small; however, this preliminary study into chronic exposure to nicotine-containing e-liquids suggests that the long-term cardiovascular risks in healthy users are limited (Polosa et al., 2017).\n\nHuman studies into the cardiovascular outcomes of nicotine consumed by vaping vary significantly in study design and rarely measure the concentrations of plasma nicotine achieved in the study participants. In addition, e-cigarette devices have evolved rapidly since first becoming commercially available in 2007 and nicotine delivery has improved in line with this evolution. Can we, or should we make a decision as to the cardiovascular effects of nicotine in e-cigarettes based on this inconclusive literature? This sentiment was clearly supported by Skotsimara et al. in their systematic review and meta-analysis (Skotsimara et al., 2019). The authors assessed all studies that investigated e-cigarettes in conjunction with cardiovascular measures, including pre-clinical (in vitro) and clinical studies. In total, 26 relevant papers were identified, out of an initial search yielding 7491 papers. When the authors carried out a meta-analysis on studies that specifically addressed the acute effects on heart rate associated with e-cigarettes, only 11 studies were eligible for inclusion, and there was significant heterogeneity between them. As expected, careful analysis showed that there was an acute increase in heart rate associated with e-cigarette use (pooled weighted MD = 2.27, 95% CI: 1.64-2.89, p <0.0001). Seven studies were analyzed for acute effects on systolic and diastolic blood pressure: “Electronic cigarette smoking significantly increased systolic blood pressure acutely (pooled weighted MD = 2.02, 95% CI: 0.07 to 3.97, p = 0.042) and diastolic blood pressure (pooled weighted MD = 2.01, 95% CI: 0.62 to 3.39, p = 0.004)”. Only three studies were analyzed for the chronic effects of switching from combustible smoking to vaping e-cigarettes on heart rate, diastolic and systolic blood pressure. No effect was observed for heart rate. “On the other hand, electronic cigarette use significantly reduced both systolic blood pressure (pooled weighted MD = –7.00, 95% CI: -9.63 to -4.37, p <0.0001) and diastolic blood pressure (pooled weighted MD = –3.65, 95% CI: -5.71 to -1.59, p = 0.001)” (Skotsimara et al., 2019).\n\nDollerup et al. found no significant differences in the time to diagnosis of ischemic heart disease or cerebrovascular disease in smokers who quit using NRT vs. no NRT at four weeks. However, after a year, there was increased diagnoses in participants using NRT (Dollerup et al., 2017). Unfortunately, this study had several limitations, which may affect the conclusions. There was no information on any other diseases the participants may have been suffering from, and therefore, the doctors prescribing NRT may have been biased towards patients who were the sickest. In addition, there were no data provided on the utilization of the NRT, only prescription records, meaning that the NRT may have not been used as intended (or at all). Not only this, but patients may have supplemented the prescription with available over-the-counter drugs or continued to smoke. No information was collected on the amount each patient was smoking before their cessation attempt, or whether they were successful. No family history was recorded, which would have alerted the authors to a predisposition to cardiovascular disease in certain patients. A meta-analysis, however, also showed that NRTs were associated with a significant increase in cardiovascular events. When these events were examined further, the authors found the association was limited to less serious events (palpitations, bradycardia and arrhythmia). There was no increased risk in major adverse cardiovascular events (cardiovascular death, nonfatal myocardial infarction, and nonfatal stroke) (Mills et al., 2014). An updated review comparing the risk of heart disease and stroke in users of either Swedish snus or American smokeless tobacco products showed there was no increase in risk found in Swedish snus users; however, there was an increase in risk of both heart disease and stroke in American smokeless tobacco product users. The authors speculate that this may be because of different levels of constituents present in the different products, but as nicotine is present in both products, it is unlikely to be the cause of the diseases observed. (Rostron et al., 2018).\n\nIn a study comparing the immediate effect of nicotine on cardiovascular parameters, transdermal nicotine patches, nicotine nasal spray and cigarette smoking were all found to increase systolic and diastolic blood pressures as well as heart rate. Cigarette smoking produced the greatest increases, followed by the nasal spray and then patches. These results indicated that the mode of nicotine delivery also has an effect on cardiovascular outcomes (Benowitz et al., 2002). In another study, no significant difference was observed in heart rate, diastolic blood pressure or systolic blood pressure in participants treated with 0, 21, 42 or 63 mg of transdermal nicotine. It should be noted that the participants were allowed to continue smoking, and so nicotine response may have already been maximal prior to patch application. If this was the case, treatment with further nicotine may not be expected to have additional effects (Zevin et al., 1998). In support of these findings, however, smokers who had suffered a major coronary event and were treated with transdermal nicotine patches showed no increased risk of suffering adverse events during a 10 week period, compared to placebo patch treatment (Joseph et al., 1996). The authors strongly concluded: “Concern about the use of nicotine replacement therapy for smokers with cardiovascular disease is not warranted” (Joseph et al., 1996). Woolf and colleagues carried out a retrospective study in patients admitted to hospital for acute cardiovascular incidences and who were discharged with a prescription for NRT. They found that NRT use was not associated with an increased risk of adverse cardiovascular events after 1 year (Woolf et al., 2012). Benowitz et al. also recently found no significant differences in the number of cardiovascular events in a group of 2022 healthy smokers who received NRT in the form of a patch, versus placebo (Benowitz et al., 2018). It should be noted that participants with pre-existing cardiovascular disorders were excluded from the study. To investigate the effect of NRT in patients with coronary heart disease, a retrospective study was carried out on 4885 smokers who were hospitalized after myocardial infarction and received NRT within two days of admission. The participants were followed until hospital discharge and monitored for readmission within a month. The authors state “[T]he use of NRT products starting during the first 2 days of hospitalization was not associated with any significant differences (harm or benefit) in outcomes among smokers hospitalized with acute coronary heart disease” (Pack et al., 2018). This further supports the evidence that there is little risk of nicotine use in the context of NRT. An ongoing clinical trial submitted in 2017 is looking to assess the cardiovascular effects of nicotine delivered in e-cigarettes over the medium term (6 months), and how this may affect use as a cessation aid (Klonizakis et al., 2017).\n\nIn contrast, a study using sublingual nicotine delivery in tablet form (4 mg) to healthy smoking participants showed that heart rate, systolic and diastolic blood pressure, rate-pressure product (heart rate*systolic blood pressure), and sympathetic nerve activity to muscle circulation (MSNA) were all significantly increased in response to nicotine compared to a placebo control. Nicotine administration showed no effect on any of the breathing parameters measured (ventilation, tidal volume, minimal oxygen saturation etc.). The authors advised that the sympathetic nerve stress caused by nicotine should be considered when prescribing the use of NRT in smokers with limited coronary reserve and those at risk of sudden hypoxic events (Najem et al., 2006). Similarly, in another study using sublingual administration of nicotine in healthy male non-smokers, administration of nicotine increased heart rate, systolic blood pressure, augmentation index (a marker of peripheral resistance) (Mitchell et al., 2005) and carotid-femoral pulse wave velocity (a marker of arterial stiffness) (Adamopoulos et al., 2009). The authors suggested that the changes in these measures were driven by an acute sympathetic response, as they were only recorded for one hour after administration. They suggest the chronic response may be different, because of the desensitization of nicotinic receptors known to occur over repeat nicotine exposures (Adamopoulos et al., 2009). This hypothesis is supported by a study carried out in healthy female smokers. Arterial stiffness was found to increase after smoking a conventional cigarette; however, no change was observed after smoking an e-cigarette. (SzołtySek-Bołdys et al., 2014). When participants were administered an acute exposure to Swedish snus, again, heart rate, diastolic and systolic blood pressure increased; however, when the data was interrogated further, it revealed that the female participants were driving this difference. There was no increase observed in the male participants. There was also no change in measures of arterial stiffness in either gender. The authors suggest that gender differences in hemodynamic responses may be responsible for this surprising result (Antoniewicz et al., 2018). A gender effect was also reported when participants were administered nicotine intravenously. Acute heart rate response at 1- and 2-minutes post infusion was significantly increased in female participants compared to their male counterparts. It is unclear whether this difference is associated with any long-term clinical outcomes (Jensen et al., 2018).\n\nA study in Parkinson’s disease patients recently found that symptoms of low blood pressure, which are common and debilitating symptoms of the disease, could be effectively treated with nicotine gum. The participants, who were all non-smokers, chewed 4 mg gum over a period of 30 minutes, which elevated both diastolic and systolic blood pressure after 10 minutes, and was sustained for 90 minutes. No serious adverse events were observed and none of the patients reached hypertensive levels, leading the authors to propose nicotine gum as a potential treatment for low blood pressure in Parkinson’s disease patients; however, their sample size consisted of only 10 individuals, and therefore, further studies are needed to confirm these findings. Studies will also need to be carried out over longer periods, to observe the development of any resistance to the effects of nicotine (DiFrancisco-Donoghue et al., 2019).\n\nAlthough there are immediate pharmacological effects of nicotine on cardiovascular parameters, there is little evidence to suggest that there is an increase in risk to long-term cardiovascular health as a result of nicotine exposure from either NRT or e-cigarettes. More importantly, e-cigarette use is substantially more favorable when compared to conventional cigarette smoking. Overall, current studies indicate that the nicotine delivered by e-cigarettes does not increase the risk of cardiovascular events in individuals who do not have any underlying cardiovascular disease.\n\n\nThrombosis, inflammation and atherosclerosis\n\nA study in 20 non-smokers showed that markers of platelet activation were increased after vaping an e-cigarette, however, the authors acknowledged “We do not know if a single constituent of the liquid mixture or the constituents as a whole could be responsible for the observed changes in platelet function” (Nocella et al., 2018). This observation was poignant in light of in vitro data, which showed that platelet activation was stimulated by e-liquid and e-liquid vapor, but suppressed by pure nicotine (Hom et al., 2016). A systematic review of three papers investigating the risk of stroke in human patients using NRT found no adverse effect; however, there was insufficient evidence to draw a definitive conclusion (Lee & Fariss, 2017). Nevertheless, in support of these findings, a study that pooled eight Swedish cohorts of male snus users also found no relationship with risk of stroke (Hansson et al., 2014).\n\nEndothelial progenitor cells (EPC) may be released from the bone marrow in response to endothelial injury. A study on 16 healthy, non-daily smokers showed that the number of EPCs was significantly increased in the blood of the participants after 10 puffs on an e-cigarette device. Levels returned to baseline within 24 hours after puffing. The authors state that this “[M]ay indicate an impact on vascular integrity leading to future atherosclerosis. However, further research is needed to understand the long-term effects of ECV” (Antoniewicz et al., 2016).\n\nNicotine has been reported to have both pro- and anti-inflammatory effects (Benowitz & Burbank, 2016). In human studies involving the use of NRTs in smoking cessation, participants have shown a reduction in inflammatory markers. It is thought that stimulation of the α7-nicotinic acetylcholine receptor (nAChR), on the macrophage cell surface leads to reduction of pro-inflammatory cytokine production, preventing sepsis (Lee & Cooke, 2011). Contrastingly, sympathetic nervous stimulation by nicotine could also induce an inflammatory response by stimulating monocyte production (Benowitz & Burbank, 2016).\n\nIn an atherosclerotic mouse model (ApoE deficient), nicotine was found to enhance atherogenesis through its angiogenic action; however, it did not initiate plaque formation, only enhanced the growth of existing plaques (Heeschen et al., 2001). Another study confirmed that short-term exposure to nicotine in mice also led to increased angiogenesis, however, chronic exposure abolished this effect. When mice were exposed to nicotine for 52 weeks, there was a 33% reduction in the number of vascular sprouts (Konishi et al., 2010). It is clear that vascular response to nicotine exposure is complex, and in human models, short-term studies may not reflect the ‘real-life’ responses to nicotine over years or even decades of intake. Differences in response to nicotine over time were also observed in thrombus formation in mice. C57BL/6 J mice were treated with 100 μg/ml in their drinking water for eight weeks (chronic exposure) and compared to an acute intravenous treatment of 3 mg/kg. No effects were observed on thrombus formation or platelet aggregation in the chronically exposed mice, however, the acute exposures caused a significant reduction in arteriole occlusion time in female mice only (Lindenblatt et al., 2007). Not only does this study add to the evidence for differences between acute and chronic exposures, but it also adds sex as a further confounding factor. Additionally, a study that chronically exposed female rats to nicotine vapor (20 hours a day, five days a week for up to two years) and produced plasma nicotine concentrations equivalent to a heavy human smoker, showed no difference in myocardial pathologies and no atherosclerotic lesions when compared to controls (Waldum et al., 1996).\n\nFahim et al. found that white blood cell, red blood cell and platelet counts, as well as hemoglobin, and hematocrit did not change when mice were treated with intraperitoneal (i.p.) nicotine (1 mg/kg) for three weeks. Despite this, pial cerebral microvessel thrombosis was significantly increased in both arterioles and venules, suggesting a higher susceptibility to cerebral thrombosis (and potentially stroke) in response to nicotine (Fahim et al., 2014).\n\nNicotine has also shown chemotactic properties, leading to accumulation of neutrophils to the endothelium (Filippini et al., 2012). Rats exposed to saline vapor containing 5% nicotine showed an increase in white blood cell count for up to 24 hours post exposure compared to saline controls. Interestingly, 10% nicotine exposure only resulted in smaller increases in white blood cell count at 6 hours post exposure, suggesting the response did not increase with dose (Ahmad et al., 2019).\n\nIn addition to sympathetic stimulation by nicotine through nAChR leading to vasoconstriction, nAChR are also expressed on endothelial cells (among other cell types). In vitro studies of endothelial cells have shown that nicotine stimulates proliferation (Villablanca, 1998), increases the expression of nitric oxide synthase (Zhang et al., 2001), and alters the production of both fibroblast growth factors and matrix metalloproteinases (Carty et al., 1996). These studies offer mechanisms by which nicotine could play a role in the development of pathological angiogenesis, atherosclerosis and platelet aggregation.\n\n\nCarcinogenesis\n\nSeveral studies have proposed mechanisms by which nicotine may activate molecular pathways leading to the propagation of tumorigenesis in vitro. Studies involving nicotine exposures in the whole organism, however, have repeatedly failed to show a significant relationship between nicotine and cancer initiation. Several documents issued by authoritative bodies also state that nicotine is not considered a carcinogen. A summary of these can be found in Table 1.\n\na The Health Consequences of Smoking—50 Years of Progress: A Report of the Surgeon General (Surgeon General, 2014)\n\nb Harmful and Potentially Harmful Constituents in Tobacco Products and Tobacco Smoke: Established List\n\nc Nicotine without smoke: Tobacco harm reduction. A report by the Tobacco Advisory Group of the Royal College of Physicians\n\nd National Academies of Sciences, Engineering, and Medicine. (2018). Public health consequences of e-cigarettes. Washington (DC), The National Academies Press\n\nAnalysis of data from the Lung Health Study assessed whether NRTs could contribute to cancer development (Murray et al., 2009). During the study, 3923 participants were randomized to receive smoking intervention, and followed over a 5-year period. Cox regression models were used to predict the contribution of NRT (nicotine gum) to the incidence of cancer occurrence observed in the participants after a 7.5-year follow-up. The use of NRT was not associated with the incidence of cancer in any of the prediction models used. There were, however, considerable limitations to the study design; for example, all the participants were previous smokers. It is impossible, therefore, to say whether any of the cancer incidences observed were because of nicotine gum or previous exposure to carcinogens from cigarettes. It could be that the effect of smoking on cancer is so strong that it eclipsed a potentially small effect from nicotine gum. In addition, the participants had been using nicotine gum for the 5-year duration of the study but had been smoking for much longer prior to that time (taken from the mean number of pack years at baseline). It may be that 5 years was not a long enough exposure to compare to the risk from smoking (Murray et al., 2009) and that the overall length of the study may not be enough time to observe manifestation of lung cancer at all. This is supported by the overall low numbers of lung cancer incidence in the cohort (n = 75).\n\nAn alternative model of nicotine exposure is snus, an oral tobacco product used predominantly in Sweden. A retrospective study was carried out in a cohort of 279,897 snus-using Swedish men, followed from 1978-1992. No increased risk was observed for either oral or lung cancer in non-smokers who used snus. There was, however, an increased risk of pancreatic cancer associated with snus use, and this was positively associated with dose (relative risk (RR): 1.9 (0.8-4.3) at 1-9 g/day; 2.1 (1.1-3.8) at ≥10 g/day) (Luo et al., 2007), although major risk factors for pancreatic cancer (alcohol use and diabetes) were not adjusted for in this study (Nilsson, 2017). It must be noted that snus it not a source of nicotine alone and contains other carcinogenic products from tobacco. Specifically, these include 4-(nitrosomethylamino)-1-(3-pyridyl)- 1-butanone (NNK), which has been shown to cause pancreatic cancer in a rat model (Rivenson et al., 1988). Therefore, NNK, for example, could have been the primary carcinogenic factor in this cohort of men, rather than nicotine. Conversely, in a pooled analysis of nine cohorts of men across Sweden, totaling 418,448 participants from 1978 to 2013, there was no increase in the incidence of pancreatic cancer found in snus users (Araghi et al., 2017). The authors concluded, “Our findings, from the largest sample to date, do not support a role of snus use in the development of pancreatic cancer in men. They, furthermore, point to tobacco smoke constituents other than nicotine or its metabolites, i.e., carcinogens associated with combustion, as the causal agent explaining the increased risk of pancreatic cancer in smokers”. Araghi et al. adjusted for both alcohol consumption and type 2 diabetes, suggesting that the observations by Luo et al. may have been due to confounding. The cohorts used in the Araghi et al. study also spanned a more recent time period. The tobacco-specific nitrosamine content of smokeless tobacco in Sweden has decreased during this time (Osterdahl et al., 2004), which would have also decreased smokeless tobacco user exposure to these carcinogens.\n\nA thorough review by Haussmann and Farriss, which analyzed studies investigating carcinogenic effect of nicotine in animal models, concluded that there was no evidence to suggest that nicotine is a complete carcinogen (i.e., causes tumor initiation, promotion and progression). When analyzing studies assessing whether nicotine could modulate carcinogenesis, there was significant variation in results. This may be because of differences in study design, or the animal models used (e.g., immunocompromised vs. competent, dose, delivery method etc.). The authors concluded that there is enough evidence to state: “[N]icotine can stimulate carcinogenesis of inoculated cancer cells in laboratory animals, especially immunocompromised mouse models”. There is insufficient evidence to demonstrate whether nicotine could modulate chemically induced cancer (Haussmann & Fariss, 2016). The overwhelming message of this review was that the data currently available is not sufficient to draw definitive conclusions. Well-designed and implemented studies, particularly in humans, are required to address the uncertainty once and for all.\n\nThe role of nicotine in cancer modulation in vivo has been supported by several in vitro studies, which elucidate nicotine-stimulated pathways leading to cellular proliferation, angiogenesis and resistance to apoptosis. A transcriptome sequencing study showed that exposing normal breast epithelial cells (MCF-10 A) to nicotine altered the expression of 138 transcripts by more than twofold. Upstream analysis and gene ontology isolated matrix metalloproteinase 2 (MMP2) as a potential candidate for promoting tumor progression after nicotine exposure (Bavarva et al., 2013). MMP2 is an enzyme that cleaves molecules involved in signal transduction, and so may have many roles in vivo. This study was preliminary, providing an unbiased approach to detecting changes to transcription. Analysis at the RNA level, however, is limited because levels of transcription are not always reflected by levels of translation and action at the protein level. Although this study provides a good list of candidates for future investigation, it is impossible to deduce what mechanisms are involved from the results thus far.\n\nA study by Nishioka et al. showed that nicotine exposure leads to the proliferation of cells that are already DNA damaged, through the overriding of the G1/S checkpoint. Human and lung epithelial cell lines were exposed to nicotine (0.5 µM) after damage had been induced by either gamma radiation or benzopyrene. Although cell proliferation was not restored to the level seen in undamaged cells, there was a significant increase in the levels of proliferation observed with the addition of nicotine, compared to the damaged cells alone. Corresponding increases in cyclins A and D1 were observed with the nicotine treatment. Checkpoint kinase 2 (Chk2) protein is phosphorylated in damaged cells leading to activation, cell cycle arrest, DNA repair or apoptosis. When damaged cells were treated with nicotine, Chk2 remained unphosphorylated. Interestingly, there seemed to be no effect of nicotine on the G2/M phase. This study shows a novel mechanism by which nicotine may act to promote the survival and proliferation of damaged cells. The experiments shown were carried out in vitro and therefore caution must be taken when extrapolating to the whole organism. If applicable in the human model, however, this mechanism could present a role for nicotine in compounding the carcinogenic effects of other tobacco smoke components. (Nishioka et al., 2011). The Surgeon General summarized this point in its 2014 report, “[T]here is substantial experimental evidence indicating that nicotine is bioactive for a number of carcinogenic mechanisms in experimental systems. Although in vitro data are suggestive of relevant biological activity, this is not supported overall by the most recent experimental animal studies” (Surgeon General, 2014).\n\n\nMetastasis\n\nThere is very limited data surrounding the role of nicotine exposure to metastasis; however, a possible effect has been found in cell lines exposed to nicotine. To investigate the increased occurrence of pancreatic cancer in smokers, a range of pancreatic cancer cell lines were used (CD18/HPAF, Capan1 and FG/Colo357) as well as non-cancerous human pancreatic ductal epithelial cells and exposed to a range of nicotine concentrations (0.1 µM, 1 µM and 5 µM). A rise in the α7 subunit of α7 nAChR was observed along with a corresponding dose-dependent upregulation of mucin 4 (MUC4) expression at both the protein and RNA levels. The increase in MUC4 could be attenuated using an α7 nAchR antagonist and led to increased migration of cells in a wound healing assay. Further downstream analysis led to the proposal of the pathway outlined in Figure 2 (Momi et al., 2013). The authors confirmed their observations in a mouse model; unfortunately, they used a cigarette smoke exposure, so the effect cannot be attributed specifically to nicotine. This study is preliminary in terms of design and cannot be extrapolated into a human model.\n\nNicotine interacts with the α7 nAChR on the cell surface, activating JAK2 and leading to the phosphorylation of STAT3. ERK1/2 mediated STAT3 phosphorylation is known to enhance DNA binding and to stabilize STAT3 in the cytoplasm. The phosphorylated forms of STAT3 join to form dimers and translocate into the nucleus where they interact with the DNA causing upregulation of MUC4. MUC4 is then expressed on the cell membrane and either directly inhibits the integration of integrins with extracellular matrix, or indirectly through HER2 activation. These pathways lead to a decrease in cell-to-cell adhesion and increase the migratory potential of pancreatic cancer cells. This figure has been reproduced with permission from Momi et al., 2013.\n\nSupporting the findings of this study, an in vitro model of head and neck cell carcinoma showed that nicotine at a concentration of 0.5 µM increased cell proliferation and viability, as well as metastatic characteristics through activation of the epidermal growth factor pathway (Shimizu et al., 2019). Only one concentration of nicotine was used, which was approximately three times higher than that achieved in the blood plasma of humans using NRT or e-cigarette devices (Kyte & Gewirtz, 2018). Although these experiments demonstrate potential pathways of nicotine action, in vivo data is required to establish whether they are relevant to the cancer in the organism.\n\n\nReproductive effects\n\nMale fertility is more sensitive to the effects of nicotine compared to female fertility (Jorsaraei et al., 2012). Nicotine concentrations of between 70 and 300 µg/L (0.43–1.85 µM) have been found in the seminal fluids of daily smokers (Oyeyipo et al., 2014), suggesting high transfer from the blood stream, and therefore, testicular cells and spermatogenesis may be vulnerable to its effects.\n\nThe primary function of sperm cells is to fertilize the oocyte, a task that is reliant on viability, motility and a successful acrosome reaction. The acrosome is an enzyme-filled capsule on the head of the sperm, which reacts to break down the zona pellucida of the oocyte on contact (Figure 3).\n\n1) Sperm cells are chemo-attracted to the oocyte and once in close proximity, interact with zona pellucida (ZP) glycoproteins. The arrangement of these glycoproteins are species specific and are responsible for preventing cross species fertilization. 2) ZP glycoproteins initiate a signaling cascade through receptor binding on the sperm surface leading to a degradation of the acrosome membrane and release of hyaluronase and acrosin, which breaks down the ZP. 3) Once through the ZP, the sperm enters the vitelline space and contacts the oocyte membrane. Binding occurs at the posterior portion of the sperm head. 4) The membranes of oocyte and sperm fuse, causing a rapid depolarization and hardening of the ZP, preventing polyspermy. The nucleus of the sperm enters the oocyte and the fertilized oocyte completes second meiosis.\n\nIn vivo studies have mostly been carried out in rat models. Daily administration of oral nicotine at 0.5 and 1.0 mg/kg body weight (considered in the range of human exposure through cigarette smoking) (Jana et al., 2010) for four weeks was found to significantly reduce the count and motility of sperm in rats in a dose-dependent manner, although there was no change in viability (Oyeyipo et al., 2011). The reduction in motility could have been because of a significant increase in the occurrence of spermatozoa with “curve tail”, a change in morphology resulting in a U shape forming in the tail. In addition to the sperm parameters tested, the rats from this study also showed a decrease in libido, shown by a reduction in mounting attempts. None of the untreated female rats housed with males from the high dose treatment group conceived during the study period, compared to 100% conception in the untreated male group. This effect was reversed after nicotine was withdrawn (Oyeyipo et al., 2011).\n\nConsistent with the observations above, daily i.p. injection of nicotine at 0.6 mg/kg body weight in rats for six weeks led to seminiferous tubule and spermatogenic derangement in the testes as well as reduced overall testicular weight (Jana et al., 2010), also observed in orally administered rats (Oyeyipo et al., 2010). These effects could be partially rescued by the human chorionic gonadotropin (stimulating androgenesis) or taurine (an antioxidant) co-supplementation (Jana et al., 2010). There was a reduction in the number of germ cells at several generations in the sperm cycle and abnormalities to sperm morphology were also significantly increased, particularly in the sperm head and formation of a “banana-like” shape. Not only were plasma testosterone levels decreased after nicotine administration, suggesting impaired androgenesis, but also luteinizing hormone (LH) and follicle stimulation hormone (FSH) levels, indicating disruption of normal hormonal release from the pituitary gland. The authors hypothesized that most of the effects shown in sperm were a result of the reduction in testosterone, caused by nicotine decreasing LH and FSH plasma concentrations (potentially by increasing glucocorticoid release from the adrenal gland), and inhibiting the production and activity of androgenic enzymes. Other contributing factors may include DNA damage and an increase in reactive oxygen species (Jana et al., 2010). Very similar findings have been shown in a recent study in adolescent rats, which were administered 0.6 mg/kg of nicotine via i.p. injection over 12 weeks (Budin et al., 2017). A higher concentration of subcutaneously injected nicotine in rats (2.5 mg/kg body weight) also resulted in smaller testes and smaller seminiferous tubule diameter, as well as reduced serum testosterone compared to untreated controls (Abd El-Aziz et al., 2016).\n\nEx vivo exposure of sperm from healthy, non-smoking men to a range of nicotine concentrations over three hours was found to decrease both the number of viable sperm and their motility. There was also an increase in the number of cells that had undergone a spontaneous acrosome reaction, disabling their ability to fertilize the oocyte. The negative effects of nicotine, however, were only found when sperm were exposed to concentrations far beyond physiological (1-10 mM). The sperm were also washed of all seminal fluids prior to exposure, so the effects seen cannot be applied to the biological environment (Oyeyipo et al., 2014). A similar experiment looked at the kinetic parameters of sperm from 10 healthy men exposed to nicotine and cotinine at multiple concentrations ex vivo. The dose that corresponded to the physiological exposure in smokers (70 ng/ml) had no effect on the kinetic parameters. There was a reduction in the kinetic properties of sperm only when a high dose (35 µg/ml) of nicotine was applied (Jorsaraei et al., 2012).\n\nResults from the studies above seem to imply a disparity between the in vivo and ex vivo effects of nicotine on male fertility. There are several reasons why this may be the case. The male humans who participated in the ex vivo studies were all non-smokers. It may be that by the time the sperm is mature and ejaculated, the cells are not as vulnerable to the effects of nicotine. They would have also had a ‘normal’ physiological environment during spermatogenesis. Not only this, but the period of exposure was much shorter (hours) compared to the in vivo models (weeks), which may not be enough time to observe an effect. In the in vivo models, the animals were exposed to nicotine for longer periods, which corresponded to multiple stages of spermatogenesis and are more reminiscent of the longer-term usage seen in ‘real-life’ smokers. In these cases, the disruption to androgenesis seemed to be key in the sub-fertility observed. We cannot, however, overlook the possibility of species differences between human and rodent models. There are no studies that investigate fertility in men exposed exclusively to nicotine. This includes a lack of observational studies in men using NRTs. Although cigarette smoking introduces considerable confounders compared to nicotine exposure alone, many studies report a negative effect of smoking on both sperm parameters (El Mulla et al., 1995; Evans et al., 1981; Shi et al., 2001; Vine et al., 1996) and in vitro fertilization outcomes (Joesbury et al., 1998; Zitzmann et al., 2003). Given the similarities between the observations in male human smokers and the nicotine-exposed rats, it may be plausible that at least some of the characteristics shown in men may be as a result of nicotine exposure. Further studies are needed to clarify this relationship.\n\n\nFemale fertility\n\nThe female reproductive system has been reported to be less sensitive to nicotine compared to the male (Riesenfeld & Oliva, 1987). Women metabolize nicotine and cotinine (the principle metabolite of nicotine) much faster compared to men (Benowitz et al., 2006). This increased metabolism is an estrogenic effect, and could offer a protective mechanism; however, the hypothesis that nicotine is associated with reduced fertility in females remains. Unfortunately, there are no studies into the effects of nicotine on fertility in women, although cotinine can be detected in the follicular fluid of women exposed to cigarette smoke (Zenzes et al., 1996), showing plausibility for an effect directly on the reproductive cells.\n\nA study in mice showed that subcutaneous injection of nicotine (5 mg/kg/day) for 30 days resulted in a significant increase in the number of apoptotic oocytes after superovulation. This effect could be rescued with co-administration of vitamin E (60 mg/kg/day orally) (Asadi et al., 2013). Vitamin E acts as an antioxidant, and therefore its effect on oocyte viability suggests that nicotine may cause an increase in oxidative stress. The dosing regimen used in this study is unclear. One would assume that the entire dose was not administered in one injection, as the lethal dose of nicotine in mice is reported to be 3.3 mg/kg (Mayer, 2014). There was no justification for the dosage used.\n\nSimilarly, i.p. injection of nicotine at 6.25 ng/g body weight in rats either twice, three or four times a day, showed a dose-dependent reduction in the number of oocytes ovulated. When animals were treated with cotinine in the same regimen, no difference was found, suggesting a nicotine-specific mechanism. The effect on ovulation was accompanied by a reduction in circulating estrogen concentrations (Blackburn et al., 1994).\n\nBordel et al. hypothesized that follicular vascularization would be altered by treatment with nicotine, because of studies suggesting the positive effect of nicotine on angiogenesis in tumorigenesis models. They transplanted follicles from donor hamsters into recipients with a dorsal skin-fold chamber, so that follicular vascularization could be observed over time in a live animal model. Angiogenesis was not changed in either of the nicotine treatment groups compared to controls, although capillary diameter was increased. Interestingly, however, follicles of the nicotine-treated animals showed higher levels of apoptosis in the granulosa cells surrounding the oocyte, and a subsequent decrease in follicular size (Bordel et al., 2005). Granulosa cells interact closely with and support the maturing oocyte, so a reduction in granulosa cell number could lead to subnormal oocyte development.\n\nIn rhesus monkeys, injection of 0.05 mg/kg nicotine resulted in alterations to fallopian tube motility that was dependent on the stage of the menstrual cycle. This could result in alterations to oocyte transit from ovary to uterus – a critical point for conception. Unfortunately, the authors could not rule out a possible confounding effect of adrenaline, which was shown to increase as a result of stress during the experimental procedure, and may have also altered the contractile characteristics of the fallopian tube (Neri & Marcus, 1972).\n\nRats treated with nicotine via transdermal patch showed no difference in the number of implanted fetuses or in integrin β3 staining of the endometrium, a marker of endometrial receptivity, compared to non-treated controls. The authors stated that the nicotine concentration achieved was equivalent to a 70 kg woman smoking 15 cigarettes per day, and were treated for three cycles before pregnancy was achieved. (Akpak et al., 2017).\n\nIt is clear that the effect of nicotine on female fertility is far from fully understood. There is a lack of consistency between animal models, dosing regimen and hypotheses, and the results do not allow any clear conclusions for the reproductive outcomes in nicotine-exposed animals. Although there are many indications of possible cellular-level alterations in response to nicotine, there is no evidence to suggest a reduction in conception rates or number of offspring, which is the primary measure of fertility. It is impossible, therefore, to begin to formulate a hypothesis in humans. This is an area where further research would be beneficial. Currently there is not enough evidence to suggest that nicotine is detrimental to female fertility.\n\n\nPregnancy and the offspring\n\nThere is a consensus that use of NRTs during pregnancy is less damaging to the fetus than smoking cigarettes, but should only be recommended if a pregnant mother is unlikely to stop smoking by any other means (Dempsey & Benowitz, 2001). Nicotine exposure is considered as detrimental to the developing fetus, however, there is very little data that examines the effects of nicotine on human fetal growth without the confounding effects of cigarette smoking at various time-points during gestation. The FDA deem nicotine as a category D drug: “For pregnancy category D, if there is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience or studies in humans, but the potential benefits from the use of the drug in pregnant women may be acceptable despite its potential risks” (Food and Drug Administration, 2008). For this reason, there is a reluctance to commission studies involving the use of NRTs in pregnant women, due to fear of any detrimental consequences. Many women, who have taken part in studies that make use of NRTs during pregnancy, also smoke concurrently (Oncken et al., 2008). There is also a high chance of misclassification because of the stigma surrounding smoking during pregnancy and the consequential likelihood of women misreporting their actual cigarette consumption (Dietz et al., 2011).\n\nOther confounding factors must also be considered when looking at nicotine intake during pregnancy. Many studies compare the risk of adverse outcomes between smoking and NRT, however, NRTs do not contain the other chemical constituents associated with cigarette smoking, and therefore risk reduction may be because of a reduction of other compounds. In addition, the nicotine itself is often delivered at much lower concentrations using NRTs compared to cigarette smoking (Bruin et al., 2010), and therefore the direct effect of nicotine on pregnancy and fetal outcomes may be masked by changes in dose. This could be a key factor when considering the use of nicotine in e-cigarettes during pregnancy, as these products may be able to deliver comparable concentrations to cigarette smoking.\n\n\nUterine blood flow\n\nA study of 35 women investigated uterine and umbilical blood flow first after smoking a single cigarette, and then, approximately two weeks later, chewing a piece of nicotine gum. Both non-smoking mothers and habitual smoking mothers were included in the study and compared. The only statistically significant effects were seen in mothers who were habitual smokers; no significant effects of either smoking or chewing nicotine-containing gum was observed in non-smokers. In the habitual smoking group, there was an increase in the flow velocity waveform systolic-to-diastolic ratio of the umbilical artery after chewing the gum, and in those who smoked more than 10 cigarettes a day, a similar effect was observed after smoking the cigarette. The authors hypothesized that the effect could be a result of more efficient smoking and therefore nicotine uptake in the habitual smokers, as well as an increased number of nicotinic receptors. However, biological markers of nicotine uptake were not measured. No effect was seen on uterine blood flow in any of the groups. Although the authors cite differences in species and study design to explain these contradictory findings, the data looks to trend towards a positive correlation. It could be that small sample numbers and high variation are masking a potential outcome (Bruner & Forouzan, 1991).\n\nIn a pilot study, six women who smoked during pregnancy were treated with a nicotine patch after 12 hours of smoking abstinence. Parameters of “fetal wellbeing” were then measured. Maternal salivary nicotine concentration increased as expected, but fetal heart rate, umbilical artery Doppler assessment and uterine contractility were not altered by the treatment. This is in contradiction to the uterine blood flow measurements found in the rat, although all the mothers in the study had been smokers throughout their pregnancies, and at treatment were already in their third trimester. Therefore, uterine blood flow may have been lower compared to non-smoking mothers. The dose of nicotine delivered was also much lower (21 mg) compared to the rat study, meaning that concentrations may be too low to produce a measurable outcome. The effects of nicotine patch treatment were only observed over a six-hour period and for one patch treatment. The effects of prolonged nicotine patch exposure were not tested (Wright et al., 1997). This could also explain a difference in outcome with results from a study looking at fetal parameters during 24-hour nicotine patch application over four days. Fetal heart rate was found to be reduced in comparison to baseline (measured a week previously during normal maternal smoking behavior) in the mornings of the 2nd, 3rd and 4th days of testing. The reduction was not considered to be clinically significant for fetal outcome (Ogburn et al., 1999). A strength of this study was that the participants were admitted to the study as inpatients, so their smoke-free status could be closely monitored; however, only 21 participants were included and the previous exposure of the fetuses to daily cigarette smoke may have affected the baseline measurements (Ogburn et al., 1999).\n\nIn pseudo pregnant rats injected with 0.5 mg/kg body weight, uterine blood flow was significantly reduced from 20–120 minutes post injection, without decreasing cardiac output. When injected with 5.0 mg/kg body weight, cardiac output and uterine blood flow were both significantly decreased. In the high dose group, uterine oxygen tension was also measured and was found to be significantly reduced. The low dose group was not measured for this parameter. The authors acknowledge that the concentrations of nicotine used in their experiment exceed the exposure obtained from smoking a cigarette, but suggest that low concentrations in the human may be enough to create a hypoxic fetal environment (Hammer et al., 1981).\n\nIn a more clinically relevant model, pregnant rats were exposed to nicotine aerosol inhalation (NAI), which resulted in blood nicotine concentrations comparable to those after human smoking. The data showed that “NAI exposure in dose and kinetics equivalent to that in human smoking stimulates autonomic nAChRs resulting in disturbances in cardiac function and systemic hemodynamics as well as vasoconstriction of the uterine artery that disrupt uteroplacental hemodynamics, which can lead to fetal ischemia”. The authors pointed out that the placenta has a large reserve capacity, which may act as a buffer to the hemodynamic effects of nicotine exposure. It is therefore unknown whether the transient depression in uterine blood flow in response to smoking a cigarette may lead to an outcome for the fetus (Shao et al., 2017).\n\n\nBirth outcomes\n\nDuring pregnancy, the placenta, in addition to its role in transferring nutrients, gas and waste to and from the fetus, acts to protect against toxicological insult. In the case of nicotine, however, higher concentrations have been found in the fetal serum, placenta and amniotic fluid compared to maternal serum concentrations (Luck et al., 1985). Although the placenta slows the uptake of nicotine to the fetus, it also slows its clearance, leading to an accumulation and higher exposure in the fetus compared to the mother (Fischer et al., 2017; Slotkin, 2008). Therefore, even in isolation, away from the additional harmful effects of cigarette smoke, the direct effect of nicotine on fetal development should be considered. The mode of nicotine delivery has also been found to lead to different levels of accumulation in the fetus, with transdermal patches leading to the highest fetal exposures (Slotkin, 2008). The authors did not examine the type of NRT used separately in this study.\n\nTwo studies used data from participants from the Danish National Birth Cohort to investigate the impact of nicotine on birth outcomes. Using NRT did not increase the risk of stillbirth when compared to stillbirths in non-smoking and non-NRT-using pregnant mothers, however, other safety outcomes (e.g., preterm birth, low birth weight etc.) were not measured (Strandberg-Larsen et al., 2008). Morales-Suárez-Varela et al. investigated the incidence of congenital malformations in NRT-using pregnant mothers. NRT use in the first 12 weeks of pregnancy was associated with a relative prevalence rate ratio of 1.61 across all congenital malformations and 2.05 for musculoskeletal malformations. Interestingly, there was a greater prevalence of malformations in mothers who used NRT compared to smokers. The authors hypothesized that this may be due to a higher number of pregnancy losses in smokers, leading to fewer live births with congenital malformations. Data on miscarriages was not included in the analysis and the conclusions were based on 19 malformations in 250 pregnancies (Morales-Suárez-Varela et al., 2006). A systematic review and meta-analysis of five randomized control trials of NRT use in pregnant women showed no significant differences in pregnancy outcomes (preterm birth, perinatal mortality, fetal deaths, neonatal intensive care admissions or miscarriage) for the women who received NRT, vs. those who did not. The authors point out that this could be as a result of non-adherence of the participants (Coleman et al., 2011).\n\nA recent study carried out in pregnant women from the UK found that NRT was not associated with an increased risk in stillbirth, supporting the Danish findings, and those of the meta-analysis described above (Dhalwani et al., 2019). It must be noted, however, that women who were recorded as sole NRT users were categorized based on prescription data, and therefore misclassification is likely. In addition, stillbirth data was recorded from primary care records, and as most women give birth in a hospital setting, there is a chance that some cases may have been missed (Dhalwani et al., 2019).\n\n\nLung development\n\nMaternal smoking during pregnancy has been shown to result in adverse lung development in the offspring (Chhabra et al., 2014). How much of this effect is due to nicotine is not known in humans, however, nicotinic receptors have been detected in several pulmonary cell types (Filippini et al., 2012) suggesting that nicotine could bind and initiate downstream signaling during development.\n\nA preliminary study into DNA methylation of aborted human fetal lungs, showed that placental cotinine concentration positively correlated with changes in several gene-specific methylation markers associated with various pathways, including asthma. However, the authors had no smoking history data for the mothers included in the study, and therefore the origin of the nicotine exposure could not be determined. Causality cannot be established from this observation, but the authors propose the use of differentially methylated regions as “a potential marker of injury in the developing lung exposed to nicotine in utero”. Further research is needed to establish the mechanisms behind lung injury in response to nicotine (Chhabra et al., 2014).\n\nThree studies from the same research group have been carried out to investigate lung development in the rhesus monkey during maternal nicotine exposure (Proskocil et al., 2005; Sekhon et al., 1999; Sekhon et al., 2001). This is a more similar model to human pregnancy in terms of placentation and uterine environment, in comparison to rodent models. Additionally, rodents are born in a more premature state compared to humans and monkeys, including the developmental stage of their lungs, making comparison even more challenging. In the first study by Sekhon et al., rhesus monkeys were dosed with nicotine at 1.0 mg/kg/day, using an osmotic pump, from day 26 of gestation until birth. Offspring were delivered by caesarean section on day 134 of gestation (equivalent to week 32 of human gestation). Levels of nicotine in the maternal serum, amniotic fluid and cord plasma were comparable to concentrations measured in pregnant human heavy smokers. Alveolar airspace was significantly increased, and corresponding surface area was decreased in nicotine-exposed offspring compared to controls. Alpha-7 nicotinic receptor subunits were identified in airway and arterial smooth muscle cells, fibroblasts surrounding the walls of airways as well as sub-mucous glands, airway-associated nerve fibers, and free pulmonary macrophages. Expression was significantly increased in cells lining the airway walls of nicotine-exposed offspring, suggesting that nicotine induced the upregulation of its receptor in the lung tissue, consistent with observations in the brain (van de Kamp & Collins, 1994). The authors hypothesized that the increase in nicotinic receptors they observed in fibroblasts could lead to an increase in collagen deposition within the lung. In a follow-up experiment, using similar methods, the authors found that both the lung weight and lung volume was significantly reduced in rhesus monkey neonates that had been exposed to nicotine in utero. In addition, they found significant reductions in some lung function parameters, such as peak tidal expiratory volume, and an increase in pulmonary resistance. These results led the authors to conclude “These findings show that nicotine exposure during gestation leads to altered pulmonary function at birth” (Sekhon et al., 2001).\n\nIn the study by Proskocil et al., offspring were delivered at day 160 of gestation (near term) by caesarean section. The lung function and histology of the offspring were measured; forced expiratory flow was significantly reduced in the nicotine-exposed group compared to controls, although forced expiratory volume was unchanged. The content of lung elastin was significantly reduced in both peripheral and central regions of the nicotine-treated offspring’s lungs; however, collagen was unchanged (contrary to the authors’ hypothesis in their previous paper). The changes shown were not mediated by any of the cytokines measured, leptin or cortisol (Proskocil et al., 2005), however, some of the effects observed could be rescued by co-administration of vitamin C.\n\nThese papers support findings from the lungs of nicotine exposures in rodents (despite the drawbacks in rodent models) (Maritz & Dennis, 1998; Wongtrakool et al., 2012) and some of the observations in the lungs of human offspring of smoking mothers (Guerra et al., 2013; Sekhon et al., 2001). Although smoking is likely to have a more profound effect on human lung development than nicotine alone, it is possible that nicotine is responsible for some of the outcomes observed; however, the mechanisms remain unclear.\n\n\nNeurological effects\n\nDuring fetal gestation, neurotransmitters act to orchestrate the development of specific neurological pathways (Levitt et al., 1997). Nicotine is a neuroactive substance, which acts through nAChRs present from neural tube formation in the embryo (Kinney et al., 1993). These induce changes to early brain morphology, however, as development continues, the morphological differences disappear. Despite the lack of long-term structural neurological changes in response to gestational nicotine exposure, there are additional effects, which are complex and difficult to map - they continue to change the trajectory of neurogenesis even when exposure has ceased. To make the picture more complex, nicotine has been shown to have both pro- and anti-apoptotic action on neurological tissue, as well as induce and prevent oxidative stress depending on the timing, cell type and concentration of exposure (Pauly & Slotkin, 2008; Zelikoff et al., 2018). Additionally, stimulation of nAChRs led to the release of several neurotransmitters, including dopamine and serotonin, so the effect of nicotine exposure may be more far reaching than the acetylcholine pathways (Pauly & Slotkin, 2008). Several studies suggest that the changes induced by nicotine in the developing brain lead to behavioral alterations that persist to adolescence and beyond (Tiesler & Heinrich, 2014). There are so many exposures that could play a role in the uterine environment, as well as post-natal exposures, in addition to the wide-ranging descriptors that cover behavioral disorders, that it is impossible to pinpoint nicotine as a direct cause.\n\nIn a study carried out in rhesus monkeys (a collaboration between Slotkin et al. and Sekhon et al., who carried out the lung development studies described above), the effects of maternal nicotine treatment on fetal brain nAChR binding, adenylyl cyclase activity and developmental biomarkers were examined (Slotkin et al., 2005). Nicotine exposure resulted in an upregulation of α4β2 nAChRs across all regions of the fetal brain, similar to the levels observed in an adult smoker. α7 nAChRs were upregulated in the occipital cortex only. For all of the other parameters tested, the results were very specific to brain regions and there was no clear overriding pattern of effect; however, in addition to the changes in receptor induction, there was evidence to suggest decreased adenylyl cyclase activity in the occipital cortex and decreased cell numbers in the caudate region. Interestingly, the authors found an interaction with nicotine and both vitamin C and choline supplementation, which was protective in some instances and detrimental in others. They comment that as choline is recommended as a supplement during pregnancy in the USA, this could offer a mechanism that puts the offspring of smoking mothers at further risk of some brain abnormalities. Overall, the authors conclude that the findings in the rhesus monkey support the observations seen in developing rodent brain models, and are also likely to translate to human development (Slotkin et al., 2005).\n\nIn a later study by the same group using the same rhesus monkey model, the effect of chronic nicotine exposure during gestation on serotonin (5-HT) in the brain was examined. Slotkin and colleagues found that levels of 5-HT were increased in in the brainstems of nicotine exposed offspring, compared to non-exposed controls (Slotkin et al., 2012). In a similar experiment in baboons, Duncan et al. found altered 5-HT binding in the medullae of nicotine exposed offspring. This altered 5-HT activity may have implications regarding the cardiorespiratory control, supported by the observation of increased heart rate variation in the exposed animals (Duncan et al., 2009).\n\nWang et al. used a newly developed ‘brain-on-a-chip’ model to investigate the effects of nicotine on human fetal brain development. Multi-cell-type brain organoids were grown in vitro and exposed to nicotine at 1 µM (similar to heavy human smoking) and 10 µM (above physiological concentration). The authors found that the 1 µM nicotine exposure led to premature neuronal differentiation increased neurite outgrowth and dysregulated forebrain and hindbrain cell populations. Although this model of human brain development is still a long way from mimicking development in the human fetus, it provides a considerable advance from traditional in vitro methods (Wang et al., 2018).\n\n\nMetabolic disorders\n\nThe effects of fetal and neonatal in utero nicotine exposure may not become apparent until later on in the life course of the offspring. Several studies in rats have described various adult outcomes including hypertension (Xiao et al., 2008), increased adiposity (Fan et al., 2016), and decreased pancreatic beta cell mass (Bruin et al., 2008) after exposure to nicotine during gestation and lactation. The mechanisms behind these associations are currently unclear, but are likely to include increased oxidative stress (Bruin et al., 2008) and could provide a link to the effects of smoking during human pregnancy to the increased prevalence of hypertension (Blake et al., 2000) and type 2 diabetes (Bruin et al., 2010). Although nicotine is a possible cause, there are no studies investigating the effects of in utero nicotine on measures of impaired cardiovascular or metabolic outcomes in humans.\n\nIn conclusion, although the use of e-cigarettes have been estimated to be up to 95% less toxic compared to conventional cigarettes (McNeill et al., 2015), and NRTs to cause very low risk of harm (Niaura, 2016), pregnancy provides a different paradigm. The weight of the evidence provided by animal studies suggests that nicotine itself is likely to be detrimental to fetal lung (Proskocil et al., 2005) and brain development, possibly at concentrations lower than the threshold needed to cause a reduction in birth weight (Slotkin et al., 2005). Long-term changes to nicotinic receptor distribution in the offspring, and the potential raft of associated neurological alterations (Pauly & Slotkin, 2008) make the downstream effects of in utero nicotine exposure broad and difficult to isolate in experimental models.\n\n\nLimitations\n\nThis narrative review was not carried out as a systematic review. Only the PubMed database was searched and therefore additional literature from other sources may have been excluded.\n\n\nConclusion\n\nLiterature investigating the effects of nicotine exposure in relation to cardiovascular risks, cancer risks and reproductive outcomes in humans is limited. As a result, it is difficult to pinpoint the role of nicotine as a single risk factor in the manifestation of these multifactorial conditions. No single risk factor can give rise to multifactorial disease by itself because, as a minimum, both the environment and endogenous risk factors must interact. Rothman and Greenland state, “[T]he importance of multicausality is that most identified causes are neither necessary nor sufficient to produce disease” (Rothman & Greenland, 2005).\n\nWhile the mechanisms by which nicotine acts on the cardiovascular system are well established and can be observed in vivo after nicotine administration, the long-term consequences of these responses are not known. Currently, the literature suggests that in consumers with no underlying cardiovascular pathology, there is no increased risk due to nicotine exposure.\n\nThere is general consensus that nicotine is not a direct or complete carcinogen, however, it remains to be established whether it has a role in cancer propagation and metastasis. Experimental molecular biology can demonstrate possible cancer progression pathways that may be brought about in response to nicotine, but these have not been investigated in cases of human cancer.\n\nFurther studies are needed to determine whether nicotine is detrimental to fertility in humans. Nevertheless, the results from animal studies indicate that nicotine has many and wide-ranging effects on fetal development.\n\nWe are now entering a period of rapid change in smoking behavior, from conventional combustible products to new ENDS, and extensive scientific research is needed to characterize the effects of nicotine exposure in humans more thoroughly.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: Extended Data - Biological Effects of Nicotine Exposure: A narrative review of the scientific literature. https://doi.org/10.6084/m9.figshare.9638234.v1 (Price & Martinez, 2019)\n\nThis project contains the following extended data:\n\nPaper Screening Master File.xlsx (Spreadsheet of full list of articles returned by search)\n\nSupplementary Methods.docx (Search methods, criteria and results for a non-systematic literature review)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors would like to thank Thilo Paschke, Paulina Andre, Kelli Sayers and Karin Jacobson for their expert assistance during the preparation of this manuscript.\n\n\nReferences\n\nAbd El-Aziz GS, El-Fark MO, Hamdy RM: Protective effect of Eruca sativa seed oil against oral nicotine induced testicular damage in rats. Tissue Cell. 2016; 48(4): 340–348. PubMed Abstract \| Publisher Full Text\n\nAdamopoulos D, Argacha JF, Gujic M, et al.: Acute effects of nicotine on arterial stiffness and wave reflection in healthy young non-smokers. Clin Exp Pharmacol Physiol. 2009; 36(8): 784–789. PubMed Abstract \| Publisher Full Text\n\nAhmad S, Zafar I, Mariappan N, et al.: Acute pulmonary effects of aerosolized nicotine. Am J Physiol Lung Cell Mol Physiol. 2019; 316(1): L94–L104. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkpak YK, Cekmez Y, Erdoğan Çakır A, et al.: An animal model of effects of nicotine exposure on endometrial receptivity and embryo implantation in pregnancy. J Matern Fetal Neonatal Med. 2017; 30(23): 2818–2823. PubMed Abstract \| Publisher Full Text\n\nAntoniewicz L, Bosson JA, Kuhl J, et al.: Electronic cigarettes increase endothelial progenitor cells in the blood of healthy volunteers. Atherosclerosis. 2016; 255: 179–185. PubMed Abstract \| Publisher Full Text\n\nAntoniewicz L, Novo M, Bosson J, et al.: Brief exposure to Swedish snus causes divergent vascular responses in healthy male and female volunteers. PLoS One. 2018; 13(4): e0195493. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAraghi M, Rosaria Galanti M, Lundberg M, et al.: Use of moist oral snuff (snus) and pancreatic cancer: Pooled analysis of nine prospective observational studies. Int J Cancer. 2017; 141(4): 687–693. PubMed Abstract \| Publisher Full Text\n\nAsadi E, Shabani R, Ghafari S, et al.: Preventing Effect of Vitamin E on Oocytes Apoptosis in Morphine-treated Mice. Int J Morphol. 2013; 31(2): 533–538. Publisher Full Text\n\nBaraona LK, Lovelace D, Daniels JL, et al.: Tobacco Harms, Nicotine Pharmacology, and Pharmacologic Tobacco Cessation Interventions for Women. J Midwifery Womens Health. 2017; 62(3): 253–269. PubMed Abstract \| Publisher Full Text\n\nBavarva JH, Tae H, Settlage RE, et al.: Characterizing the Genetic Basis for Nicotine Induced Cancer Development: A Transcriptome Sequencing Study. PLoS One. 2013; 8(6): e67252. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBenowitz NL, Lessovschlaggar CN, Swan GE, et al.: Female sex and oral contraceptive use accelerate nicotine metabolism. Clin Pharmacol Ther. 2006; 79(5): 480–488. PubMed Abstract \| Publisher Full Text\n\nBenowitz NL: Pharmacology of nicotine: addiction and therapeutics. Annu Rev Pharmacol Toxicol. 1996; 36(1): 597–613. PubMed Abstract \| Publisher Full Text\n\nBenowitz NL, Society for Research on Nicotine and Tobacco: Nicotine Safety and Toxicity. Oxford University Press. 1998. Reference Source\n\nBenowitz NL, Burbank AD: Cardiovascular toxicity of nicotine: Implications for electronic cigarette use. Trends Cardiovasc Med. 2016; 26(6): 515–523. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBenowitz NL, Pipe A, West R, et al.: Cardiovascular Safety of Varenicline, Bupropion, and Nicotine Patch in Smokers: A Randomized Clinical Trial. JAMA Intern Med. 2018; 178(5): 622–631. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBenowitz NL, Hansson A, Jacob P 3rd: Cardiovascular effects of nasal and transdermal nicotine and cigarette smoking. Hypertension. 2002; 39(6): 1107–12. PubMed Abstract \| Publisher Full Text\n\nBlackburn CW, Peterson CA, Hales HA, et al.: Nicotine, but not cotinine, has a direct toxic effect on ovarian function in the immature gonadotropin-stimulated rat. Reprod Toxicol. 1994; 8(4): 325–331. PubMed Abstract \| Publisher Full Text\n\nBlake KV, Gurrin LC, Evans SF, et al.: Maternal cigarette smoking during pregnancy, low birth weight and subsequent blood pressure in early childhood. Early Hum Dev. 2000; 57(2): 137–47. PubMed Abstract \| Publisher Full Text\n\nBordel R, Laschke MW, Menger MD, et al.: Nicotine does not affect vascularization but inhibits growth of freely transplanted ovarian follicles by inducing granulosa cell apoptosis. Hum Reprod. 2005; 21(3): 610–617. PubMed Abstract \| Publisher Full Text\n\nBruin JE, Gerstein HC, Holloway AC: Long-term consequences of fetal and neonatal nicotine exposure: a critical review. Toxicol Sci. 2010; 116(2): 364–374. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBruin JE, Petre MA, Lehman MA, et al.: Maternal nicotine exposure increases oxidative stress in the offspring. Free Radic Biol Med. 2008; 44(11): 1919–1925. PubMed Abstract \| Publisher Full Text\n\nBruner JP, Forouzan I: Smoking and buccally administered nicotine. Acute effect on uterine and umbilical artery Doppler flow velocity waveforms. J Reprod Med. 1991; 36(6): 435–40. PubMed Abstract\n\nBudin SB, Kho JH, Lee JH, et al.: Low-dose Nicotine Exposure Induced the Oxidative Damage of Reproductive Organs and Altered the Sperm Characteristics of Adolescent Male Rats. Malays J Med Sci. 2017; 24(6): 50–57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCameron JM, Howell DN, White JR, et al.: Variable and potentially fatal amounts of nicotine in e-cigarette nicotine solutions. Tob Control. 2014; 23(1): 77–78. PubMed Abstract \| Publisher Full Text\n\nCarnevale R, Sciarretta S, Violi F, et al.: Acute Impact of Tobacco vs Electronic Cigarette Smoking on Oxidative Stress and Vascular Function. Chest. 2016; 150(3): 606–612. PubMed Abstract \| Publisher Full Text\n\nCarty CS, Soloway PD, Kayastha S, et al.: Nicotine and cotinine stimulate secretion of basic fibroblast growth factor and affect expression of matrix metalloproteinases in cultured human smooth muscle cells. J Vasc Surg. 1996; 24(6): 927–34; discussion 934–5. PubMed Abstract \| Publisher Full Text\n\nChatham-Stephens K, Law R, Taylor E, et al.: Exposure Calls to U. S. Poison Centers Involving Electronic Cigarettes and Conventional Cigarettes-September 2010-December 2014. J Med Toxicol. 2016; 12(4): 350–357. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChaumont M, de Becker B, Zaher W, et al.: Differential Effects of E-Cigarette on Microvascular Endothelial Function, Arterial Stiffness and Oxidative Stress: A Randomized Crossover Trial. Sci Rep. 2018; 8(1): 10378. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChhabra D, Sharma S, Kho AT, et al.: Fetal lung and placental methylation is associated with in utero nicotine exposure. Epigenetics. 2014; 9(11): 1473–1484. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nColeman T, Chamberlain C, Cooper S, et al.: Efficacy and safety of nicotine replacement therapy for smoking cessation in pregnancy: systematic review and meta-analysis. Addiction. 2011; 106(1): 52–61. PubMed Abstract \| Publisher Full Text\n\nD’Ruiz CD, O’Connell G, Graff DW, et al.: Measurement of cardiovascular and pulmonary function endpoints and other physiological effects following partial or complete substitution of cigarettes with electronic cigarettes in adult smokers. Regul Toxicol Pharmacol. 2017; 87: 36–53. PubMed Abstract \| Publisher Full Text\n\nDempsey DA, Benowitz NL: Risks and benefits of nicotine to aid smoking cessation in pregnancy. Drug Safety. 2001; 24(4): 277–322. PubMed Abstract \| Publisher Full Text\n\nDhalwani NN, Szatkowski L, Coleman T, et al.: Stillbirth Among Women Prescribed Nicotine Replacement Therapy in Pregnancy: Analysis of a Large UK Pregnancy Cohort. Nicotine Tob Res. 2019; 21(4): 409–415. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDietz PM, Homa D, England LJ, et al.: Estimates of nondisclosure of cigarette smoking among pregnant and nonpregnant women of reproductive age in the United States. Am J Epidemiol. 2011; 173(3): 355–359. PubMed Abstract \| Publisher Full Text\n\nDiFrancisco-Donoghue J, Jung MK, Leder A: Nicotine Gum as a Therapeutic Approach for Low Blood Pressure in Parkinson's Disease: A Randomized Pilot Study. Nicotine Tob Res. 2019; 21(2): 253–256. PubMed Abstract \| Publisher Full Text\n\nDollerup J, Vestbo J, Murray-Thomas T, et al.: Cardiovascular risks in smokers treated with nicotine replacement therapy: a historical cohort study. Clin Epidemiol. 2017; 9: 231–243. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDuncan JR, Garland M, Myers MM, et al.: Prenatal nicotine-exposure alters fetal autonomic activity and medullary neurotransmitter receptors: implications for sudden infant death syndrome. J Appl Physiol (1985). 2009; 107(5): 1579–1590. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEl Mulla KF, Köhn FM, El Beheiry AH, et al.: The effect of smoking and varicocele on human sperm acrosin activity and acrosome reaction. Hum Reprod. 1995; 10(12): 3190–4. PubMed Abstract \| Publisher Full Text\n\nEvans HJ, Fletcher J, Torrance M, et al.: Sperm abnormalities and cigarette smoking. Lancet. 1981; 1(8221): 627–9. PubMed Abstract \| Publisher Full Text\n\nFahim MA, Nemmar A, Al-Salam S, et al.: Thromboembolic injury and systemic toxicity induced by nicotine in mice. Gen Physiol Biophys. 2014; 33(3): 345–355. PubMed Abstract \| Publisher Full Text\n\nFan J, Zhang W, Rao Y, et al.: Perinatal Nicotine Exposure Increases Obesity Susceptibility in Adult Male Rat Offspring by Altering Early Adipogenesis. Endocrinology. 2016; 157(11): 4276–4286. PubMed Abstract \| Publisher Full Text\n\nFarsalinos K, Cibella F, Caponnetto P, et al.: Effect of continuous smoking reduction and abstinence on blood pressure and heart rate in smokers switching to electronic cigarettes. Intern Emerg Med. 2016; 11(1): 85–94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFilippini P, Cesario A, Fini M, et al.: The Yin and Yang of non-neuronal α7-nicotinic receptors in inflammation and autoimmunity. Curr Drug Targets. 2012; 13(5): 644–55. PubMed Abstract \| Publisher Full Text\n\nFischer ST, Lili LN, Li S, et al.: Low-level maternal exposure to nicotine associates with significant metabolic perturbations in second-trimester amniotic fluid. Environ Int. 2017; 107: 227–234. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFood and Drug Administration: Proposed Rules on current pregnancy, labor and delivery, and lactation labeling - Federal Register, 73 §. 2008.\n\nFranke FE, Thomas JE: The Treatment of Acute Nicotine Poisoning. JAMA. 1936; 106(7): 507–512. Publisher Full Text\n\nFranzen KF, Willig J, Cayo Talavera S, et al.: E-cigarettes and cigarettes worsen peripheral and central hemodynamics as well as arterial stiffness: A randomized, double-blinded pilot study. Vasc Med. 2018; 23(5): 419–425. PubMed Abstract \| Publisher Full Text\n\nGehlbach SH, Williams WA, Perry LD, et al.: Green-tobacco sickness. An illness of tobacco harvesters. JAMA. 1974; 229(14): 1880–3. PubMed Abstract \| Publisher Full Text\n\nSurgeon General: The Health Consequences of Smoking—50 Years of Progress: A Report of the Surgeon General. 2014. Reference Source\n\nGottlieb S, Zeller M: A Nicotine-Focused Framework for Public Health. N Engl J Med. 2017; 377(12): 1111–1114. PubMed Abstract \| Publisher Full Text\n\nGuerra S, Stern DA, Zhou M, et al.: Combined effects of parental and active smoking on early lung function deficits: a prospective study from birth to age 26 years. Thorax. 2013; 68(11): 1021–1028. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaass M, Kübler W: Nicotine and sympathetic neurotransmission. Cardiovasc Drugs Ther. 1997; 10(6): 657–665. PubMed Abstract \| Publisher Full Text\n\nHammer RE, Goldman H, Mitchell JA: Effects of nicotine on uterine blood flow and intrauterine oxygen tension in the rat. J Reprod Fertil. 1981; 63(1): 163–8. PubMed Abstract \| Publisher Full Text\n\nHansson J, Galanti MR, Hergens MP, et al.: Snus (Swedish smokeless tobacco) use and risk of stroke: pooled analyses of incidence and survival. J Intern Med. 2014; 276(1): 87–95. PubMed Abstract \| Publisher Full Text\n\nHaussmann HJ, Fariss MW: Comprehensive review of epidemiological and animal studies on the potential carcinogenic effects of nicotine per se. Crit Rev Toxicol. 2016; 46(8): 701–734. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeeschen C, Jang JJ, Weis M, et al.: Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis. Nat Med. 2001; 7(7): 833–839. PubMed Abstract \| Publisher Full Text\n\nHom S, Chen L, Wang T, et al.: Platelet activation, adhesion, inflammation, and aggregation potential are altered in the presence of electronic cigarette extracts of variable nicotine concentrations. Platelets. 2016; 27(7): 694–702. PubMed Abstract \| Publisher Full Text\n\nIkonomidis I, Vlastos D, Kourea K, et al.: Electronic Cigarette Smoking Increases Arterial Stiffness and Oxidative Stress to a Lesser Extent Than a Single Conventional Cigarette: An Acute and Chronic Study. Circulation. 2018; 137(3): 303–306. PubMed Abstract \| Publisher Full Text\n\nJana K, Samanta PK, De DK: Nicotine diminishes testicular gametogenesis, steroidogenesis, and steroidogenic acute regulatory protein expression in adult albino rats: possible influence on pituitary gonadotropins and alteration of testicular antioxidant status. Toxicol Sci. 2010; 116(2): 647–659. PubMed Abstract \| Publisher Full Text\n\nJensen KP, Valentine G, Buta E, et al.: Biochemical, demographic, and self-reported tobacco-related predictors of the acute heart rate response to nicotine in smokers. Pharmacol Biochem Behav. 2018; 173: 36–43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJoesbury KA, Edirisinghe WR, Phillips MR, et al.: Evidence that male smoking affects the likelihood of a pregnancy following IVF treatment: application of the modified cumulative embryo score. Hum Reprod. 1998; 13(6): 1506–1513. PubMed Abstract \| Publisher Full Text\n\nJohnston R, Crowe M, Doma K: Effect of nicotine on repeated bouts of anaerobic exercise in nicotine naïve individuals. Eur J Appl Physiol. 2018; 118(4): 681–689. PubMed Abstract \| Publisher Full Text\n\nJorsaraei SGA, Jorsaraei SGA, Shibahara H: The in-vitro effects of nicotine, cotinine and leptin on sperm parameters analyzed by CASA system. Int J Reprod Biomed. 2012; 6.\n\nJoseph AM, Norman SM, Ferry LH, et al.: The safety of transdermal nicotine as an aid to smoking cessation in patients with cardiac disease. N Engl J Med. 1996; 335(24): 1792–1798. PubMed Abstract \| Publisher Full Text\n\nKinney HC, O’Donnell TJ, Kriger P, et al.: Early developmental changes in [3H]nicotine binding in the human brainstem. Neuroscience. 1993; 55(4): 1127–38. PubMed Abstract \| Publisher Full Text\n\nKlonizakis M, Crank H, Gumber A, et al.: Smokers making a quit attempt using e-cigarettes with or without nicotine or prescription nicotine replacement therapy: Impact on cardiovascular function (ISME-NRT) - a study protocol. BMC Public Health. 2017; 17(1): 293. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKonishi H, Wu J, Cooke JP: Chronic exposure to nicotine impairs cholinergic angiogenesis. Vasc Med. 2010; 15(1): 47–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKyte SL, Gewirtz DA: The Influence of Nicotine on Lung Tumor Growth, Cancer Chemotherapy, and Chemotherapy-Induced Peripheral Neuropathy. J Pharmacol Exp Ther. 2018; 366(2): 303–313. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee J, Cooke JP: The role of nicotine in the pathogenesis of atherosclerosis. Atherosclerosis. 2011; 215(2): 281–283. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee PN, Fariss MW: A systematic review of possible serious adverse health effects of nicotine replacement therapy. Arch Toxicol. 2017; 91(4): 1565–1594. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLevitt P, Harvey JA, Friedman E, et al.: New evidence for neurotransmitter influences on brain development. Trends Neurosci. 1997; 20(6): 269–274. PubMed Abstract \| Publisher Full Text\n\nLindenblatt N, Platz U, Hameister J, et al.: Distinct effects of acute and chronic nicotine application on microvascular thrombus formation and endothelial function in male and female mice. Langenbecks Arch Surg. 2007; 392(3): 285–295. PubMed Abstract \| Publisher Full Text\n\nLuck W, Nau H, Hansen R, et al.: Extent of nicotine and cotinine transfer to the human fetus, placenta and amniotic fluid of smoking mothers. Dev Pharmacol Ther. 1985; 8(6): 384–95. PubMed Abstract \| Publisher Full Text\n\nLuo J, Ye W, Zendehdel K, et al.: Oral use of Swedish moist snuff (snus) and risk for cancer of the mouth, lung, and pancreas in male construction workers: a retrospective cohort study. Lancet. 2007; 369(9578): 2015–2020. PubMed Abstract \| Publisher Full Text\n\nMaritz GS, Dennis H: Maternal nicotine exposure during gestation and lactation interferes with alveolar development in the neonatal lung. Reprod Fertil Dev. 1998; 10(3): 255–61. PubMed Abstract \| Publisher Full Text\n\nMayer B: How much nicotine kills a human? Tracing back the generally accepted lethal dose to dubious self-experiments in the nineteenth century. Arch Toxicol. 2014; 88(1): 5–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcneill A, Brose LS, Calder R, et al.: E-cigarettes: an evidence update A report commissioned by Public Health England. 2015. Reference Source\n\nMills EJ, Thorlund K, Eapen S, et al.: Cardiovascular events associated with smoking cessation pharmacotherapies: a network meta-analysis. Circulation. 2014; 129(1): 28–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMishra A, Chaturvedi P, Datta S, et al.: Harmful effects of nicotine. Indian J Med Paediatr Oncol. 2015; 36(1): 24–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMitchell GF, Lacourcière Y, Arnold JM, et al.: Changes in aortic stiffness and augmentation index after acute converting enzyme or vasopeptidase inhibition. Hypertension. 2005; 46(5): 1111–1117. PubMed Abstract \| Publisher Full Text\n\nMoheimani RS, Bhetraratana M, Peters KM, et al.: Sympathomimetic Effects of Acute E-Cigarette Use: Role of Nicotine and Non-Nicotine Constituents. J Am Heart Assoc. 2017a; 6(9): pii: e006579. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoheimani RS, Bhetraratana M, Yin F, et al.: Increased Cardiac Sympathetic Activity and Oxidative Stress in Habitual Electronic Cigarette Users: Implications for Cardiovascular Risk. JAMA Cardiol. 2017b; 2(3): 278–284. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMomi N, Ponnusamy MP, Kaur S, et al.: Nicotine/cigarette smoke promotes metastasis of pancreatic cancer through α7nAChR-mediated MUC4 upregulation. Oncogene. 2013; 32(11): 1384–1395. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMorales-Suárez-Varela MM, Bille C, Christensen K, et al.: Smoking habits, nicotine use, and congenital malformations. Obstet Gynecol. 2006; 107(1): 51–57. PubMed Abstract \| Publisher Full Text\n\nMowry JB, Spyker DA, Brooks DE, et al.: 2014 Annual Report of the American Association of Poison Control Centers' National Poison Data System (NPDS): 32nd Annual Report. Clin Toxicol (Phila). 2015; 53(10): 962–1147. PubMed Abstract \| Publisher Full Text\n\nMurray RP, Connett JE, Zapawa LM: Does nicotine replacement therapy cause cancer? Evidence from the Lung Health Study. Nicotine Tob Res. 2009; 11(9): 1076–1082. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNajem B, Houssière A, Pathak A, et al.: Acute cardiovascular and sympathetic effects of nicotine replacement therapy. Hypertension. 2006; 47(6): 1162–7. PubMed Abstract \| Publisher Full Text\n\nNeri A, Marcus SL: Effect of nicotine on the motility of the oviducts in the rhesus monkey: a preliminary report. J Reprod Fertil. 1972; 31(1): 91–7. PubMed Abstract \| Publisher Full Text\n\nNiaura R: Re-Thinking Nicotine and its Effects, 1–24. 2016. Reference Source\n\nNilsson R: Use of rodent data for cancer risk assessment of smokeless tobacco in the regulatory context. Regul Toxicol Pharmacol. 2017; 88: 338–348. PubMed Abstract \| Publisher Full Text\n\nNishioka T, Yamamoto D, Zhu T, et al.: Nicotine overrides DNA damage-induced G1/S restriction in lung cells. PLoS One. 2011; 6(4): e18619. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNocella C, Biondi-Zoccai G, Sciarretta S, et al.: Impact of Tobacco Versus Electronic Cigarette Smoking on Platelet Function. Am J Cardiol. 2018; 122(9): 1477–1481. PubMed Abstract \| Publisher Full Text\n\nOgburn PL Jr, Hurt RD, Croghan IT, et al.: Nicotine patch use in pregnant smokers: nicotine and cotinine levels and fetal effects. Am J Obstet Gynecol. 1999; 181(3): 736–743. PubMed Abstract \| Publisher Full Text\n\nOncken C, Dornelas E, Greene J, et al.: Nicotine gum for pregnant smokers: a randomized controlled trial. Obstet Gynecol. 2008; 112(4): 859–867. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOsterdahl BG, Jansson C, Paccou A: Decreased levels of tobacco-specific N-nitrosamines in moist snuff on the Swedish market. J Agric Food Chem. 2004; 52(16): 5085–5088. PubMed Abstract \| Publisher Full Text\n\nOyeyipo IP, Maartens PJ, du Plessis SS: In vitro effects of nicotine on human spermatozoa. Andrologia. 2014; 46(8): 887–892. PubMed Abstract \| Publisher Full Text\n\nOyeyipo IP, Raji Y, Emikpe BO, et al.: Effects of oral administration of nicotine on organ weight, serum testosterone level and testicular histology in adult male rats. Niger J Physiol Sci. 2010; 25(1): 81–86. PubMed Abstract\n\nOyeyipo IP, Raji Y, Emikpe BO, et al.: Effects of nicotine on sperm characteristics and fertility profile in adult male rats: a possible role of cessation. J Reprod Infertil. 2011; 12(3): 201–7. PubMed Abstract \| Free Full Text\n\nPack QR, Priya A, Lagu TC, et al.: Short-Term Safety of Nicotine Replacement in Smokers Hospitalized With Coronary Heart Disease. J Am Heart Assoc. 2018; 7(18): e009424. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaik JH, Kang S, Durey A, et al.: Symptomatic bradycardia due to nicotine intoxication. Rev Bras Ter Intensiva. 2018; 30(1): 121–126. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPauly JR, Slotkin TA: Maternal tobacco smoking, nicotine replacement and neurobehavioural development. Acta Paediatr. 2008; 97(10): 1331–1337. PubMed Abstract \| Publisher Full Text\n\nPolosa R, Cibella F, Caponnetto P, et al.: Health impact of E-cigarettes: a prospective 3.5-year study of regular daily users who have never smoked. Sci Rep. 2017; 7(1): 13825. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrice L, Martinez J: Extended Data - Biological Effects of Nicotine Exposure: A narrative review of the scientific literature. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9638234.v1\n\nProskocil BJ, Sekhon HS, Clark JA, et al.: Vitamin C prevents the effects of prenatal nicotine on pulmonary function in newborn monkeys. Am J Respir Crit Care Med. 2005; 171(9): 1032–1039. PubMed Abstract \| Publisher Full Text\n\nRiesenfeld A, Oliva H: The effect of nicotine and alcohol on the fertility and life span of rats. A cytological analysis. Acta Anat (Basel). 1987; 128(1): 45–50. PubMed Abstract \| Publisher Full Text\n\nRivenson A, Hoffmann D, Prokopczyk B, et al.: Induction of lung and exocrine pancreas tumors in F344 rats by tobacco-specific and Areca-derived N-nitrosamines. Cancer Res. 1988; 48(23): 6912–7. PubMed Abstract\n\nRostron BL, Chang JT, Anic GM, et al.: Smokeless tobacco use and circulatory disease risk: a systematic review and meta-analysis. Open Heart. 2018; 5(2): e000846. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRothman KJ, Greenland S: Causation and causal inference in epidemiology. Am J Public Health. 2005; 95 Suppl 1: S144–150. PubMed Abstract \| Publisher Full Text\n\nRoyal College of Physicians: Nicotine without smoke: Tobacco harm reduction. 2016. Reference Source\n\nSabha M, Tanus-Santos JE, Toledo JC, et al.: Transdermal nicotine mimics the smoking-induced endothelial dysfunction. Clin Pharmacol Ther. 2000; 68(2): 167–174. PubMed Abstract \| Publisher Full Text\n\nSaleeon T, Siriwong W, Maldonado-Perez HL, et al.: Salivary cotinine levels as a biomarker for green tobacco sickness in dry tobacco production among Thai traditional tobacco farmers. Rocz Panstw Zakl Hig. 2016; 67(2): 121–130. PubMed Abstract\n\nSekhon HS, Jia Y, Raab R, et al.: Prenatal nicotine increases pulmonary alpha7 nicotinic receptor expression and alters fetal lung development in monkeys. J Clin Invest. 1999; 103(5): 637–647. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSekhon HS, Keller JA, Benowitz NL, et al.: Prenatal nicotine exposure alters pulmonary function in newborn rhesus monkeys. Am J Respir Crit Care Med. 2001; 164(6): 989–994. PubMed Abstract \| Publisher Full Text\n\nShao XM, López-Valdés HE, Liang J, et al.: Inhaled nicotine equivalent to cigarette smoking disrupts systemic and uterine hemodynamics and induces cardiac arrhythmia in pregnant rats. Sci Rep. 2017; 7(1): 16974. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShi Q, Ko E, Barclay L, et al.: Cigarette smoking and aneuploidy in human sperm. Mol Reprod Dev. 2001; 59(4): 417–421. PubMed Abstract \| Publisher Full Text\n\nShimizu R, Ibaragi S, Eguchi T, et al.: Nicotine promotes lymph node metastasis and cetuximab resistance in head and neck squamous cell carcinoma. Int J Oncol. 2019; 54(1): 283–294. PubMed Abstract \| Publisher Full Text\n\nSkotsimara G, Antonopoulos AS, Oikonomou E, et al.: Cardiovascular effects of electronic cigarettes: A systematic review and meta-analysis. Eur J Prev Cardiol. 2019; 26(11): 1219–1228. PubMed Abstract \| Publisher Full Text\n\nSlotkin TA: If nicotine is a developmental neurotoxicant in animal studies, dare we recommend nicotine replacement therapy in pregnant women and adolescents? Neurotoxicol Teratol. 2008; 30(1): 1–19. PubMed Abstract \| Publisher Full Text\n\nSlotkin TA, Seidler FJ, Qiao D, et al.: Effects of prenatal nicotine exposure on primate brain development and attempted amelioration with supplemental choline or vitamin C: neurotransmitter receptors, cell signaling and cell development biomarkers in fetal brain regions of rhesus monkeys. Neuropsychopharmacology. 2005; 30(1): 129–144. PubMed Abstract \| Publisher Full Text\n\nSlotkin TA, Seidler FJ, Spindel ER: Prenatal nicotine exposure in rhesus monkeys compromises development of brainstem and cardiac monoamine pathways involved in perinatal adaptation and sudden infant death syndrome: amelioration by vitamin C. Neurotoxicol Teratol. 2012; 33(3): 431–434. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSolarino B, Rosenbaum F, Riesselmann B, et al.: Death due to ingestion of nicotine-containing solution: case report and review of the literature. Forensic Sci Int. 2010; 195(1–3): e19–22. PubMed Abstract \| Publisher Full Text\n\nStrandberg-Larsen K, Tinggaard M, Nybo Andersen AM, et al.: Use of nicotine replacement therapy during pregnancy and stillbirth: a cohort study. BJOG. 2008; 115(11): 1405–1410. PubMed Abstract \| Publisher Full Text\n\nSzołtySek-Bołdys I, Sobczak A, Zielińska-Danch W, et al.: Influence of inhaled nicotine source on arterial stiffness Zmiana sztywności tętnic w zależności od źródła inhalowanej nikotyny. Przegląd Lekarski. 2014; (11): 572–575. Reference Source\n\nTiesler CM, Heinrich J: Prenatal nicotine exposure and child behavioural problems. Eur Child Adolesc Psychiatry. 2014; 23(10): 913–929. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan de Kamp JL, Collins AC: Prenatal nicotine alters nicotinic receptor development in the mouse brain. Pharmacol Biochem Behav. 1994; 47(4): 889–900. PubMed Abstract \| Publisher Full Text\n\nVansickel AR, Weaver MF, Eissenberg T: Clinical laboratory assessment of the abuse liability of an electronic cigarette. Addiction. 2012; 107(8): 1493–1500. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVardavas CI, Girvalaki C, Filippidis FT, et al.: Characteristics and outcomes of e-cigarette exposure incidents reported to 10 European Poison Centers: a retrospective data analysis. Tob Induc Dis. 2017; 15: 36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVillablanca AC: Nicotine stimulates DNA synthesis and proliferation in vascular endothelial cells in vitro. J Appl Physiol (1985). 1998; 84(6): 2089–98. PubMed Abstract \| Publisher Full Text\n\nVine MF, Tse CK, Hu P, et al.: Cigarette smoking and semen quality. Fertil Steril. 1996; 65(4): 835–42. PubMed Abstract \| Publisher Full Text\n\nVlachopoulos C, Ioakeimidis N, Abdelrasoul M, et al.: Electronic Cigarette Smoking Increases Aortic Stiffness and Blood Pressure in Young Smokers. J Am Coll Cardiol. 2016; 67(23): 2802–2803. PubMed Abstract \| Publisher Full Text\n\nWaldum HL, Nilsen OG, Nilsen T, et al.: Long-term effects of inhaled nicotine. Life Sci. 1996; 58(16): 1339–1346. PubMed Abstract \| Publisher Full Text\n\nWang Y, Wang L, Zhu Y, et al.: Human brain organoid-on-a-chip to model prenatal nicotine exposure. Lab Chip. 2018; 18(6): 851–860. PubMed Abstract \| Publisher Full Text\n\nWest R: Tobacco smoking: Health impact, prevalence, correlates and interventions. Psychol Health. 2017; 32(8): 1018–1036. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWongtrakool C, Wang N, Hyde DM, et al.: Prenatal nicotine exposure alters lung function and airway geometry through α7 nicotinic receptors. Am J Respir Cell Mol Biol. 2012; 45(5): 695–702. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWoolf KJ, Zabad MN, Post JM, et al.: Effect of nicotine replacement therapy on cardiovascular outcomes after acute coronary syndromes. Am J Cardiol. 2012; 110(7): 968–970. PubMed Abstract \| Publisher Full Text\n\nWright LN, Thorp JM Jr, Kuller JA, et al.: Transdermal nicotine replacement in pregnancy: maternal pharmacokinetics and fetal effects. Am J Obstet Gynecol. 1997; 176(5): 1090–1094. PubMed Abstract \| Publisher Full Text\n\nXiao D, Xu Z, Huang X, et al.: Prenatal gender-related nicotine exposure increases blood pressure response to angiotensin II in adult offspring. Hypertension. 2008; 51(4): 1239–1247. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYan XS, D’Ruiz C: Effects of using electronic cigarettes on nicotine delivery and cardiovascular function in comparison with regular cigarettes. Regul Toxicol Pharmacol. 2015; 71(1): 24–34. PubMed Abstract \| Publisher Full Text\n\nZelikoff JT, Parmalee NL, Corbett K, et al.: Microglia Activation and Gene Expression Alteration of Neurotrophins in the Hippocampus Following Early-Life Exposure to E-Cigarette Aerosols in a Murine Model. Toxicol Sci. 2018; 162(1): 276–286. PubMed Abstract \| Publisher Full Text\n\nZenzes MT, Reed TE, Wang P, et al.: Cotinine, a major metabolite of nicotine, is detectable in follicular fluids of passive smokers in in vitro fertilization therapy. Fertil Steril. 1996; 66(4): 614–619. PubMed Abstract \| Publisher Full Text\n\nZevin S, Jacob P 3rd, Benowitz NL: Dose-related cardiovascular and endocrine effects of transdermal nicotine. Clin Pharmacol Ther. 1998; 64(1): 87–95. PubMed Abstract \| Publisher Full Text\n\nZhang S, Day I, Ye S: Nicotine induced changes in gene expression by human coronary artery endothelial cells. Atherosclerosis. 2001; 154(2): 277–83. PubMed Abstract \| Publisher Full Text\n\nZitzmann M, Rolf C, Nordhoff V, et al.: Male smokers have a decreased success rate for in vitro fertilization and intracytoplasmic sperm injection. Fertil Steril. 2003; 79 Suppl 3: 1550–1554. PubMed Abstract \| Publisher Full Text" }	[ { "id": "53455", "date": "30 Sep 2019", "name": "Lukasz Antoniewicz", "expertise": [ "Reviewer Expertise Cardiovascular Research. Effects of nicotine", "e-cigarettes", "tobacco and snus on endothelial function." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis narrative review is interestingly written. However, several major points need to be addressed prior to acceptance.\n\nThe title \"Biological effects of nicotine exposure“ is misleading as the article is focusing on cardiovascular, carcinogenic and reproductive effects of nicotine. The wide spectrum of neurological effects and especially addictive effects is not mentioned or discussed.\n\nMethods: The biggest methodological issues are the inclusion criteria for this review. As stated, ten search terms were used in PubMed to carry out literature search. Additionally, relevant studies and studies prior to 2009 referenced in the returned papers were included. Additional relevant studies from JTI databases were also added. In the Supplementary Methods flowchart, initially 2301 references were found. 33 were finally included from this source. However, 31 papers were included by reference and 58 from the JTI-database. The majority of cited papers were included without standardized search criteria.\n\nIntroduction:\nThe authors state that “It is already understood that nicotine is not a major contributing factor to the diseases associated with tobacco smoking“ referring to (Benowitz et al., 1998; Gottlieb & Zeller, 2017; Royal College of Physicians, 2016). Gottlieb et al. is a short commentary, itself not referring to any data supporting this statement. It states however \"Nicotine is, however, responsible for getting smokers addicted to cigarette smoking in the first place - usually while they are children or young adults and their brains are still developing - and keeping them addicted long-term\". As mentioned before, this is omitted in the entire work.\nBenowitz et al. is a very good review on nicotine research published 1998. A more recent publication would be feasible.\nThe Royal College of Physicians states \"The safety of NRT products, which have typically been used for days or weeks in the context of an attempt to quit smoking, is well established …. with no evidence of any increase in the risk of heart attack, stroke or death\". The introduction, stating that \"it is already understood that nicotine is not a major contributing factor” is in my opinion not supported by the referenced articles.\n\n“Extensive analysis into nicotine as a pharmacological molecule has already been carried out”. In the listed references, West et al. and Baraona et al. did not carry out any investigations on nicotine as a pharmacological molecule.\n\nAcute nicotine toxicity The authors compare the ingested dose of 60 mg to the transdermally applied dose of 64 mg. The line “(interestingly higher than the “lethal” dose, although not ingested)” is obsolete as the routes of nicotine administration lead to completely different serum-nicotine-levels.\n\nCardiovascular effects In the end the authors state “Although there are immediate pharmacological effects of nicotine on cardiovascular parameters, there is little evidence to suggest that there is an increase in risk to long-term cardiovascular health as a result of nicotine exposure from either NRT or e-cigarettes.” That final statement may clearly be misread as no long-term studies (longer than 10 years of sole e-cigarette or NRT use) exist. In several paragraphs the authors discuss the effects of Swedish snus – to discuss long-term nicotine effects. Indeed several important studies suggest that long-term snus use may be associated with higher mortality when developing cardiovascular disease, the development of diabetes and endothelial dysfunction1,2,3,4,5. Especially the study by Arefalk et al. (2014)3 needs to be addressed.\n\nThrombosis, inflammation and atherosclerosis The role of nicotine in the propagation of atherosclerosis and diabetic retinopathy should be discussed more thoroughly6,7,8,9. Additionally, a summary of this topic should be added at the end of this paragraph.\n\nPregnancy and offspring Effects of snus use and pregnancy should be discussed as well. For example:10.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [ { "c_id": "4942", "date": "01 Oct 2019", "name": "Leonie Price", "role": "Author Response", "response": "Dear Dr. Antoniewicz,Thank you very much for taking the time to review our paper. We appreciate all your comments and will be more than happy to address them in the coming days, making amendments to the manuscript as appropriate. We strongly believe that paper will be significantly improved thanks to your input.Kind regards,Leonie Price and Javier Martinez" }, { "c_id": "5120", "date": "09 Jan 2020", "name": "Leonie Price", "role": "Author Response", "response": "Dear Dr. Antoniewicz, Thank you once again for taking the time to review our paper. We were advised to wait until our second peer review before making any changes to the manuscript, hence the delay in response. Please find below our reply to your suggestions point-by-point. We hope you find our changes satisfactory. Best regards, Leonie Price and Javier Martinez Point-by-point response The title \"Biological effects of nicotine exposure” is misleading as the article is focusing on cardiovascular, carcinogenic and reproductive effects of nicotine. The wide spectrum of neurological effects and especially addictive effects is not mentioned or discussed. Well noted, the title has been updated to reflect the specific areas covered in the review. Page 1 Methods: The biggest methodological issues are the inclusion criteria for this review. As stated, ten search terms were used in PubMed to carry out literature search. Additionally, relevant studies and studies prior to 2009 referenced in the returned papers were included. Additional relevant studies from JTI databases were also added. In the Supplementary Methods flowchart, initially 2301 references were found. 33 were finally included from this source. However, 31 papers were included by reference and 58 from the JTI-database. The majority of cited papers were included without standardized search criteria. We agree – We found the publications list returned from the PubMed search disappointing, especially due to the large number of relevant and important papers we had available in our databases. We felt it necessary to include these publications despite them not appearing in the PubMed list. We hope to have now addressed your concern by making this point clearer in the manuscript through expanding the methods and limitations sections. Lines 37-42 and 1047-1052 Introduction: The authors state that “It is already understood that nicotine is not a major contributing factor to the diseases associated with tobacco smoking” referring to (Benowitz et al., 1998; Gottlieb & Zeller, 2017; Royal College of Physicians, 2016). Gottlieb et al. is a short commentary, itself not referring to any data supporting this statement. It states however \"Nicotine is, however, responsible for getting smokers addicted to cigarette smoking in the first place - usually while they are children or young adults and their brains are still developing - and keeping them addicted long-term\". As mentioned before, this is omitted in the entire work. You are correct, the addictive nature of nicotine was deliberately omitted from the review as the focus was on the specific areas biological effect outlined. The secondary effects of nicotine addiction, contributing to prolonged smoking and the associated health implications are related to the effects of inhaled combusted tobacco, and do not manifest as a result of nicotine exposure alone. Having said this, we have now updated the wording of this sentence. It now reads as follows: “Various reviews of published scientific literature and Public Health statements have concluded that despite its addictive nature, nicotine is not a major contributing factor to diseases associated with tobacco smoking (Benowitz et al., 2016; Gottlieb & Zeller, 2017; Royal College of Physicians, 2016; Surgeon General, 2014)”. Lines 5-10 Benowitz et al. is a very good review on nicotine research published 1998. A more recent publication would be feasible. This citation has been updated. Line 9 The Royal College of Physicians states \"The safety of NRT products, which have typically been used for days or weeks in the context of an attempt to quit smoking, is well established …. with no evidence of any increase in the risk of heart attack, stroke or death\". The introduction, stating that \"it is already understood that nicotine is not a major contributing factor” is in my opinion not supported by the referenced articles. Addressed as part of Introduction point 1. The sentence now reads “Various reviews of published scientific literature and Public Health statements have concluded that despite its addictive nature, nicotine is not a major contributing factor to diseases associated with tobacco smoking (Benowitz et al., 2016; Gottlieb & Zeller, 2017; Royal College of Physicians, 2016; Surgeon General, 2014)”. The Royal College of Physicians do indeed state as quoted; however, the overall conclusions of their publication say the following: “People smoke because they are addicted to nicotine, but are harmed by other constituents of tobacco smoke. Provision of the nicotine that smokers are addicted to without the harmful components of tobacco smoke can prevent most of the harm from smoking”. Lines 5-10 “Extensive analysis into nicotine as a pharmacological molecule has already been carried out”. In the listed references, West et al. and Baraona et al. did not carry out any investigations on nicotine as a pharmacological molecule. We have made it clear that the Barona and West articles review the research and did not carry out the studies. Line 19. Acute nicotine toxicity The authors compare the ingested dose of 60 mg to the transdermally applied dose of 64 mg. The line “(interestingly higher than the “lethal” dose, although not ingested)” is obsolete as the routes of nicotine administration lead to completely different serum-nicotine-levels. We have deleted this sentence. Line 71 Cardiovascular effects In the end the authors state “Although there are immediate pharmacological effects of nicotine on cardiovascular parameters, there is little evidence to suggest that there is an increase in risk to long-term cardiovascular health as a result of nicotine exposure from either NRT or e-cigarettes.” That final statement may clearly be misread as no long-term studies (longer than 10 years of sole e-cigarette or NRT use) exist. In several paragraphs the authors discuss the effects of Swedish snus – to discuss long-term nicotine effects. Indeed several important studies suggest that long-term snus use may be associated with higher mortality when developing cardiovascular disease, the development of diabetes and endothelial dysfunction1,2,3,4,5. Especially the study by Arefalk et al. (2014) needs to be addressed. We understand how our wording could be misinterpreted here and have added clarity to this section. Lines 349-354 The publications by Arefalk et al. (2012), Arefalk et al. (2014), Skaug et al. (2016) have been addressed. Lines 260-274 and 191-199. Hergens et al. 2008 has been addressed alongside the publications referring to stroke risk already included, in the Thrombosis section. Lines 367-371 The suggested paper by Östenson et al. has not been included, as diabetes risk is beyond the scope of the paper. We agree that this could be an important topic for future work. None of the additional papers change the conclusions reached in the review as published. Thrombosis, inflammation and atherosclerosis The role of nicotine in the propagation of atherosclerosis and diabetic retinopathy should be discussed more thoroughly 6,7,8,9. Additionally, a summary of this topic should be added at the end of this paragraph. Thank you – Zainalabidin et al. and Li et al. have now been addressed in this section. Lines 437-455 and 466-472 We have not addressed Boretsky et al. as diabetes is beyond the scope of the review. As above, we agree that this could be an important topic for future work. We have also not included Zhao et al., as this study investigated nicotine as a potential protective substance against preeclampsia in vitro using HUVEC cells. Published studies in humans have suggested that despite a protective effect of cigarette smoking on the risk of developing preeclampsia in pregnancy, the effect is lost in pregnant women who use snus (Wikström et al., 2010; England et al., 2003). Therefore, the current hypotheses suggest that it is a substance other than nicotine in cigarette smoke that is responsible for the protective effect described epidemiologically. It is unclear, therefore, whether the in vitro findings of Zhao’s study offer any further insight into the clinical outcomes in humans. The section has been summarised. Lines 473-483 Pregnancy and offspring Effects of snus use and pregnancy should be discussed as well. For example:10 Agree. We have included the suggested reference as well as England et al. 2003. Lines 897-911" } ] }, { "id": "56498", "date": "03 Dec 2019", "name": "Bernd Mayer", "expertise": [ "Reviewer Expertise Active research in cardiovascular pharmacology", "longstanding interest in nicotine." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a comprehensive review of the most prominent and widely discussed biological effects of nicotine that could be relevant for users of new nicotine products, in particular, electronic cigarettes and tobacco heating systems. The article is well balanced, covering the relevant literature, and urgently needed to distinguish the effects of nicotine from the well established detrimental effects of tobacco smoke. I highly recommend the indexing of the paper, but the authors may wish to address the following points to further increase the impact of their work.\n\nGeneral\nPress releases on publications describing potential consequences of vaping, with or without nicotine, tend to confuse media representatives, policymakers and the public by mixing up human data from clinical studies with results from rodents exposed to vapor and in vitro data. I appreciate the authors' efforts to clearly distinguish between these categories, which differ largely in their relevance for consumers. Although the current paper describes the discrepancies between animal and human studies on several occasions, I missed a few paragraphs explicitly discussing the striking lack of translation of rodent results to humans with respect to the biology of nicotine. Such cautionary notes may help to prevent the widespread over interpretation of experimental results that are irrelevant for human consumers.\n\nWhile the harmful effects of nicotine are comprehensively discussed, the authors do not even mention potential beneficial effects, such as protection from the development of inflammatory brain diseases and ulcerative colitis as well as improvement of cognitive funtions and AD(H)S. Although controversial, these benefits of nicotine appear to be better documented than some of the harms that are extensively described in the article.\nSpecific Points\n\nAcute nicotine toxicity\nThe authors should provide the suggested value of the revised lethal oral nicotine dose for adults (0.5 - 1g; ref. Mayer (2014)1). In the paragraph on the toxicity of patches, it is stated \"although not ingested\". This is misleading because the dermal bioavailability of nicotine is much higher than oral.\n\nIt should be mentioned that the number of calls counted by poison centers does not necessarily reflect the number of incidences of real poisoning. I suggest providing the numbers concerning toilet cleaners (or varenicline) for comparison.\nCardiovascular effects\nThe cardiovascular effects of nicotine may be partially due to noradrenaline release from sympathetic nerve terminals (as illustrated in Fig. 1), but activation of nicotinic receptors expressed in vegetative ganglia should also be mentioned.\n\nThere is no compelling evidence for an increased blood pressure of smokers (statement on p. 5, left panel, 2nd paragraph). Most epidemiological studies indicate either no difference or lower blood pressure of smokers as compared to non-smokers. For relatively recent studies see e.g.: Akbarour et al. (2019)2; Li et al. (2017)3; Papathanasiou et al. (2015)4; Linneberg et al. (2015)5; Li et al. (2010)6.\n\nThe part on endothelial function should be updated by including two very recent studies on the effects of vaping with nicotine on flow-mediated dilation. While Kuntic et al. (2019)7 reported on acute endothelial dysfunction of smokers 15 minutes after vaping, George et al. (2019)8 observed significant improvement of FMD within one month of switching from smoking to electronic cigarettes.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "5121", "date": "09 Jan 2020", "name": "Leonie Price", "role": "Author Response", "response": "Dear Dr. Mayer, Thank you for taking the time to peer review our paper and provide such positive feedback. Please find below our response to your general and specific suggestions point-by-point. We hope you find our changes satisfactory. Best regards, Leonie Price and Javier Martinez General points Press releases on publications describing potential consequences of vaping, with or without nicotine, tend to confuse media representatives, policymakers and the public by mixing up human data from clinical studies with results from rodents exposed to vapor and in vitro data. I appreciate the authors' efforts to clearly distinguish between these categories, which differ largely in their relevance for consumers. Although the current paper describes the discrepancies between animal and human studies on several occasions, I missed a few paragraphs explicitly discussing the striking lack of translation of rodent results to humans with respect to the biology of nicotine. Such cautionary notes may help to prevent the widespread over interpretation of experimental results that are irrelevant for human consumers. We agree; however, we have not added any additional content regarding issues related to animal models of human disease. We hope that by dividing the manuscript into sections addressing the various in vivo and in vitro studies as well as describing differences between them enough to highlight the limitations. Unfortunately, inaccurate media representation of scientific data is an issue that spans all scientific fields. We hope that the more scientists engage with journalists, the quicker this problem can be eliminated. While the harmful effects of nicotine are comprehensively discussed, the authors do not even mention potential beneficial effects, such as protection from the development of inflammatory brain diseases and ulcerative colitis as well as improvement of cognitive functions and AD(H)S. Although controversial, these benefits of nicotine appear to be better documented than some of the harms that are extensively described in the article. We have added reference to ulcerative colitis in the context of inflammation. As you rightly point out, the positive effects of nicotine are controversial, and therefore need to be assessed with caution. We have not addressed any of the cognitive effects, as this subject falls beyond the scope of the paper; however, this could be an important topic for future work. Specific Points Acute nicotine toxicity The authors should provide the suggested value of the revised lethal oral nicotine dose for adults (0.5 - 1g; ref. Mayer (2014)1). In the paragraph on the toxicity of patches, it is stated \"although not ingested\". This is misleading because the dermal bioavailability of nicotine is much higher than oral. Thank you – we agree this was not clear and have deleted this sentence. Line 71 It should be mentioned that the number of calls counted by poison centers does not necessarily reflect the number of incidences of real poisoning. I suggest providing the numbers concerning toilet cleaners (or varenicline) for comparison. Thank you for highlighting this. We have added some context to the poison statistics in the form of data from the same US poison centers used in the Chatham-Stephens study, as well as adding your excellent point. Lines 85-92 Cardiovascular effects The cardiovascular effects of nicotine may be partially due to noradrenaline release from sympathetic nerve terminals (as illustrated in Fig. 1), but activation of nicotinic receptors expressed in vegetative ganglia should also be mentioned. Agree – we have now mentioned this mechanism. Lines 98-100 There is no compelling evidence for an increased blood pressure of smokers (statement on p. 5, left panel, 2nd paragraph). Most epidemiological studies indicate either no difference or lower blood pressure of smokers as compared to non-smokers. For relatively recent studies see e.g.: Akbarour et al. (2019)2; Li et al. (2017)3; Papathanasiou et al. (2015)4; Linneberg et al. (2015)5; Li et al. (2010)6. You are, of course, correct. The wording of this paragraph has been updated to more accurately describe the participants in the Farsalinos study, rather than smokers in general. Lines 151-153 The part on endothelial function should be updated by including two very recent studies on the effects of vaping with nicotine on flow-mediated dilation. While Kuntic et al. (2019)7 reported on acute endothelial dysfunction of smokers 15 minutes after vaping, George et al. (2019)8 observed significant improvement of FMD within one month of switching from smoking to electronic cigarettes. These references have now been added. Lines 381-403" } ] } ]	1	https://f1000research.com/articles/8-1586
https://f1000research.com/articles/9-9/v1	09 Jan 20	{ "type": "Case Report", "title": "Case Report: Pseudoxanthoma elasticum", "authors": [ "Catarina Lucas", "João Aranha", "Isabel da Rocha", "Domingos Sousa", "João Aranha", "Isabel da Rocha", "Domingos Sousa" ], "abstract": "Pseudoxanthoma elasticum (PXE) is a rare inherited disorder, characterised by a progressive mineralization and fragmentation of elastic fibres of the skin, retina and cardiovascular system. At an initial stage, the skin usually exhibits distinctive lesions and subsequently extra-dermal manifestations. The diagnosis is based on clinical manifestations, histological analysis of the lesions and genetic analysis. This is a case report of a 12-year-old child complaining of painless, mildly itchy yellow papules in the cervical region with 1 year of evolution. PXE is currently an incurable disease and has a favourable prognosis when cardiovascular and retinal complications are prevented and monitored.", "keywords": [ "Pseudoxanthoma elasticum", "ABCC6 gene", "retina angioid streaks", "hypertension" ], "content": "Introduction\n\nPseudoxanthoma elasticum (PXE), also known as Grönblad-Strandberg syndrome, is a rare inherited disorder presenting an autosomal recessive inheritance with a mutation in the ABCC6 gene (ATP-binding cassette transporter C6), mapped in the chromosome 16. It has an estimated prevalence of 1/25.000 to 1/100.000 inhabitants and is 10 times more prevalent in women1,2.\n\nThe disease is characterised by a progressive mineralization and fragmentation of elastic fibres of the skin, retina, gastrointestinal and cardiovascular systems.\n\nPXE results in a variety of signs and symptoms that vary in their number, type, and severity from person to person. Certain effects of PXE can cause serious medical problems, while others have less impact. Effects may include: skin changes, changes in the retina of the eye that may result in significant loss of central vision, changes in the cardiovascular system that may involve calcification of arteries and decreased blood flow in the arms and legs, and/or changes in the gastrointestinal system that may lead to bleeding in the stomach or intestines. At present, there is no way to predict the exact progression of the disorder for an individual2. Some people have no skin lesions; others have no vision loss. Many people do not experience gastrointestinal complications or cardiovascular difficulties. A few have no manifestations of PXE except for a positive skin biopsy or irregular streaks resembling a blood vessel (angioid) in the retina of the eye1,2.\n\nThe diagnosis is based on major and minor criteria defined in 1994 taking into account the clinical manifestations described above (Table 1), positive histological on Von Kossa stain for reticular dermis, family history and genetic analysis of the ABCC6 gene1–3.\n\nPXE is currently an incurable disease but has a favourable prognosis with appropriate follow-up by multidisciplinary teams.\n\n\nCase description\n\nA 12-year-old Caucasian girl presented for a dermatology appointment in November 2016 due to sporadically painless, slightly itchy yellow papules in the cervical region with 1 year of evolution. These lesions remained stable throughout this period with no medication or treatment. The child had no pain complaints, or inflammatory signs on the lesions or around them, and did not have any associated symptoms. Previously she was a healthy child, was not taking any daily medication and had no relevant personal or family medical history of dermatosis.\n\nOn examination, painless, uneven, rough and yellow plaques without inflammatory signs in the posterior cervical region that merged bilaterally and symmetrically into the right and left side cervical region was found (Figure 1). It was similar to a goose bump pattern, giving it a parchment-like skin appearance. The remaining integument had no changes.\n\nPhotographs of the skin lesion taken from (a) behind, (b) the left and (c) right side of the patient’s neck. Uneven, rough and yellow plaques can be seen in the cervical region.\n\nNo other abnormalities were found on the rest of physical examination.\n\nIn this appointment, the following exams were conducted: hemogram, general biochemistry with lipid profile and phosphorus-calcium balance and urinary sediment that were unremarkable. Moreover, a skin biopsy was promptly performed.\n\nIn the next appointment, still in November, the skin biopsy results revealed limited changes to the superficial/medium reticular dermis with a long strip of elastic fibre fragmentation. They were thick, granular, basophilic, with bizarre shapes between the collagen fibres with a normal appearance. There was no evidence of mineralization or deposits of mucin.\n\nFollowing the investigation, further exams were conducted: electrocardiogram and echocardiogram, carotid and aortoiliac venous and arterial ultrasonography, retinogram with full ophthalmological examination (angioid streaks). The results were normal. There was the possibility of a genetic disorder, but the pathology specialist decided there was no need to perform a study of the ABCC6 genes based on the clinical aspect of the lesions, which were highly characteristic of PXE.\n\nThe child has been having annual follow-up with ophthalmologic, paediatric cardiology and dermatology appointments. Until February 2019 no systemic manifestations were reported.\n\n\nDiscussion\n\nPXE is a rare inherited disorder, characterised by mineralization disturbances of the connective tissue with elastic fibre degeneration, mainly involving skin, eyeballs and cardiovascular system1–4.\n\nAt the consensus conference, in 1994, the PXE diagnosis criteria were defined (Table 1)3. The patient presented two of these major criteria: characteristic yellow cobblestone lesion in the skin and positive von Kossa stain.\n\nAlthough the cutaneous manifestation is frequently flesh-colored to yellow macules or papules which progressively agglutinate into bigger plaques, and the affected skin that becomes lax and wrinkled, the clinical manifestation is variable and can occur at any age. At an early stage, the skin lesions are found on the right, left and rear sides of the neck and flexion creases, including armpits, inguinal region, popliteus region, and periumbilical area, as seen in this patient. The lesions may also affect the genital area and oral mucosa2,4,5.\n\nPXE ophthalmological manifestations include mainly angioid streaks due to Bruch membrane lesions (characteristic but not pathognomonic), deformed macular degeneration, retina pigmentation, choroidal neovascularization, haemorrhage and scar formation on the retina. For the ocular complications, vascular endothelial growth factor antagonists have been used to prevent neovascularization, thus reducing the occurrence of the most severe complication: loss of vision4,6.\n\nCardiovascular manifestations are a major cause of morbidity in these patients: hypertension, angina pectoris and intermittent claudication are some of the examples. Patients with PXE may also develop early atherosclerosis due to the mineralization of the internal elastic lamina of the blood vessels and lipid alteration with high density lipoprotein (HDL) cholesterol levels reduction in blood plasma and hypertriglyceridemia. This contributes to a higher incidence of acute myocardial infarction and cerebrovascular accident6.\n\nHistopathology exams in PXE patients reveal agglutination and mineralization of elastic fibres and fragmentation of the elastic fibres in the medium/deep dermis2,7.\n\nPXE and papillary dermal elastosis share similar lesions. Therefore, a differential diagnosis shall be performed. At a histopathology level, papillary dermal elastosis with the Von Kossa stain is negative to calcium in elastic fibres. Moreover, there is no systemic involvement in papillary dermal elastosis7.\n\nThe patient’s general practitioner shall be responsible for early detection of the ocular and cardiovascular complications of the PXE because the mineralization in the centre of the fibres predisposes the development of secondary hypertension. Therefore, the morbidity and mortality caused by the disease can be monitored and reduced.\n\nTo conclude, PXE is a rare disease, the recognition of which is important since it can cause systemic severe cardiovascular and retinal manifestations. The diagnosis is based on histology result. Patients can be further tested for a mutation in the ABCC6 gene. The earlier the diagnosis, the sooner preventive measures and close observation can be adopted to prevent and control the possible adverse events caused by this disease.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the parent of the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nChristen-Zäch S, Huber M, Struck B, et al.: Pseudoxanthoma elasticum: evaluation of diagnostic criteria based on molecular data. Br J Dermatol. 2006; 155(1): 89–93. PubMed Abstract \| Publisher Full Text\n\nMarconi B, Bobyr I, Campanati A, et al.: Pseudoxanthoma elasticum and skin: Clinical manifestations, histopathology, pathomechanism, perspectives of treatment. Intractable Rare Dis Res. 2015; 4(3): 113–122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLebwohl M, Neldner K, Pope FM, et al.: Classification of pseudoxanthoma elasticum: Report of a consensus conference. J Am Acad Dermatol. 1994; 30(1): 103–107. PubMed Abstract \| Publisher Full Text\n\nRoach ES, Islam MP: Pseudoxanthoma elasticum. Handb Clin Neurol. 2015; 132: 215–221. PubMed Abstract \| Publisher Full Text\n\nRibeiro CP, Abuawad YG, Swiczar BCC, et al.: Pseudoxanthoma elasticum-like papillary dermal elastolysis. An Bras Dermatol. 2017; 92(6): 897–898. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHosen MJ, Lamoen A, De Paepe A, et al.: Histopathology of pseudoxanthoma elasticum and related disorders: histological hallmarks and diagnostic clues. Scientifica (Cairo). 2012; 2012: 598262. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLopes L, Reis MD, Gouveia AI, et al.: Elastólise da derme papilar semelhante a pseudoxantoma elástico. Rev Soc Port Dermatol Venereol. 2014; 72: 223–6. Reference Source" }	[ { "id": "58434", "date": "17 Feb 2020", "name": "Olivier M. Vanakker", "expertise": [ "Reviewer Expertise Ectopic mineralization diseases" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report describes a classic presentation of PXE in a young girl. I have a few comments on the current manuscript:\nThe manuscript refers in the introduction and discussion to the detection of a mutation in ABCC6, but PXE is an autosomal recessive disease, so this should be mutations in ABCC6.\n\nPXE can also be caused by mutations in the ENPP1 gene. This should be added.\n\nThe difference between women and man is approximately 3:1, not 10:1\n\nIn the description of the ocular lesions, there should be a distinction between the angioid streaks due to Bruch's membrane abnormalities - which are almost always present - and retinal changes, i.e. a retinal dystrophy which can occur but is much more rare.\n\nThe idea of not analyzing ABCC6 when the histology is typical is not correct for two reasons: a. in its initial presentation - both clinical and histological - PXE can high resemble the PXE-like syndrome with coagulation factor deficiency due to GGCX mutations. The natural history and follow-up of these patients is however completely different. b. identification of the causal mutations - either in ABCC6 or ENPP1 - will allow to detect subclinical patients in the family (as said, some have no skin lesions and angioid streaks do not have to become symptomatic immediately) as well as heterozygous carriers. The latter also have a higher cardiovascular risk and a higher risk for stroke and are candidates for primary prevention. So every patient should have molecular analysis performed!\n\nIn the first paragraph of the discussion, the authors mention the eyeballs. But there is nothing wrong with the eyeballs themselves.\n\nPXE patients have media calcification in their arteries, which leads to arteriosclerosis, not atherosclerosis (which reflects intima calcification).\n\nThe differential diagnosis is absolutely not complete: one should exclude the hemoglobinopathy-associated phenocopy, the PXE-like syndrome with coagulation factor deficiency, the PXE-like phenotype with pigmented retinopathy, ...\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "66211", "date": "17 Jul 2020", "name": "Márta Medvecz", "expertise": [ "Reviewer Expertise Genodermatoses" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript for a case report presents the well-documented case of a 12-year-old girl with PXE. In addition, the discussion section of the paper also includes a short review on PXE, highlighting systemic manifestations, histopathologic alterations, and management. However, there are some minor issues to be clarified by the authors:\nA more informative title could be useful, e.g. one that implies that an interesting aspect of this case is that early skin lesions in a young patient are documented.\n\nIt would raise the quality of the manuscript if, in addition to the description of the skin biopsy findings, histopathologic images could also be included. Also, for the histology, it should be mentioned, which stainings were performed.\n\nThe necessity of genetic testing should be decided by a clinical geneticist and not a pathologist, as implied in the Case description of the manuscript.\n\nIn the Discussion section, it can be debated if the patients’ general practitioner should be responsible for the early detection of systemic manifestation. PXE should be managed by a multidisciplinary team at specialized rare disease centers with sufficient expertise and experience.\n\nCertain papers cited by the manuscript are outdated or are not available in English. These should be replaced by citing more recent, high-quality publications.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "66222", "date": "20 Jul 2020", "name": "Annamaria Offidani", "expertise": [ "Reviewer Expertise Immunological skin diseases", "psoriasis", "atopic dermatitis", "genetic and rare disease of the skin" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title is written in SEO terms.\n\nThe text is clear and well written, the case is described in detail. Our response is to approve the manuscript as it doesn't present flaws or incomplete descriptions of the steps from diagnosis to management.\n\nIt would be nice if the authors could add some details of dermoscopic characteristic features they saw as dermoscopy could substitute histopathology for early and confirmed diagnosis.1\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "66220", "date": "31 Jul 2020", "name": "Wilko Spiering", "expertise": [ "Reviewer Expertise Vascular medicine", "pseudoxanthoma elasticum", "hypertension." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report reports on a 12 year old child with PXE.\n\nI have the following comments:\nSome references used are outdated and even contains a non-English paper. Please update and replace.\n\nThe male-female ratio is not correct. PXE is 2-3 times more prevalent in women.\n\nThe diagnostic criteria used are from 1994. I would recommend using the updated criteria from Plomp et al. (http://www.ncbi.nlm.nih.gov/pubmed/20358627), published in 2010.1\n\nThe authors state that PXE ‘has a favourable prognosis with appropriate follow-up by multidisciplinary teams’. What is meant by favourable? Although recommended, it is not proven that multidisciplinary teams improve any outcome in PXE.\n\nI disagree with the decision of the pathology specialist that there was no need to perform a genetic analysis for mutations in ABCC6, or other genes like ENPP1 and GCCX. A genetic analysis always should be done. This obviously cannot be changed in the manuscript, but maybe the authors can make a statement on this.\n\nEyeballs are not affected in PXE. Please change into Bruch’s membrane of the eye or retina.\n\nI would prefer to use vascular manifestations instead of cardiovascular as the heart is usually not more affected compared with healthy controls. Also, hypertension is probably not more prevalent in PXE.\n\nIt is stated that there is a ‘higher incidence of acute myocardial infarction and cerebrovascular accident in PXE’. The reference (Hosen et al.) used for this statement is a histopathology paper and does not even discuss this. Cerebrovascular accidents, but not myocardial infarctions, are indeed increased in PXE. Please use an appropriate reference.\n\nIt is stated that ‘Patients with PXE may also develop early atherosclerosis due to the mineralization of the internal elastic lamina of the blood vessels and lipid alteration with high density lipoprotein (HDL) cholesterol levels reduction in blood plasma and hypertriglyceridemia.’ Atherosclerosis is not more common in PXE, as this is an intimal disease with accumulation of cholesterol in the intima. The appropriate vascular manifestation in PXE is arteriosclerosis, with calcification of the internal elastic layer and media of the vessel wall. The lipid alterations described are pathophysiological not correct and should be deleted.\n\nRecent developments on potential treatments in PXE are lacking, e.g. etidronate (Kranenburg et al. 2018) and oral pyrophosphate (Väärämäki et al. 2019).1,2 I would recommend adding this to the discussion.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-9
https://f1000research.com/articles/8-321/v1	22 Mar 19	{ "type": "Research Article", "title": "DNA interference by a mesophilic Argonaute protein, CbcAgo", "authors": [ "Nieves García-Quintans", "Laurie Bowden", "José Berenguer", "Mario Mencía", "Nieves García-Quintans", "Laurie Bowden" ], "abstract": "Background: The search for putative enzymes that can facilitate gene editing has recently focused its attention on Argonaute proteins from prokaryotes (pAgos). Though they are structural homologues of human Argonaute protein, which uses RNA guides to interfere with RNA targets, pAgos use ssDNA guides to identify and, in many cases, cut a complementary DNA target. Thermophilic pAgos from Thermus thermophilus, Pyrococcus furiosus and Methanocaldococcus jasmanii have been identified and thoroughly studied, but their thermoactivity makes them of little use in mesophilic systems such as mammalian cells. Methods: Here we search for and identify CbcAgo, a prokaryotic Argonaute protein from a mesophilic bacterium, and characterize in vitro its DNA interference activity. Results: CbcAgo efficiently uses 5’P-ssDNA guides as small as 11-mers to cut ssDNA targets, requires divalent cations (preferentially, Mn2+) and has a maximum activity between 37 and 42 °C, remaining active up to 55 °C. Nicking activity on supercoiled dsDNA was shown. However, no efficient double-strand breaking activity could be demonstrated. Conclusions: CbcAgo can use gDNA guides as small as 11 nucleotides long to cut complementary ssDNA targets at 37ºC, making it a promising starting point for the development of new gene editing tools for mammalian cells.", "keywords": [ "Argonaute", "prokaryotic", "mesophilic", "gene edition", "characterization", "DNA-DNA interference" ], "content": "Introduction\n\nArgonaute proteins play a central role in gene silencing and defense against external RNA in eukaryotes, binding small RNA molecules that are then used as guides to scan for complementary RNA targets in the form of mRNAs or RNA viruses. Depending on the presence of specific residues in the protein sequence, the targets can be cut or simply blocked, with degradation carried out by other proteins, leading to inhibition of the expression (silencing) of the genes involved1. The structure of eukaryotic Argonaute proteins (eAgo) consists of four domains organized in a specific order (N-PAZ-MID-PIWI) which are each involved in different steps of the protein’s enzyme activity.\n\nHomologues to eAgo that contain these four domains are found in both bacteria and archaea, being collectively known as prokaryotic Argonaute (pAgo) proteins. Their function seems to depend on the species, targeting either RNA or DNA2. The best studied pAgos, such as those from Thermus thermophilus (ThAgo)3, Pyrococcus furiosus (PfAgo)4 and Methanocaldococcus jannaschii (MjAgo)5, use ssDNA guides (gDNA) to target DNA in vitro, with ThAgo and PfAgo shown to be involved in defense against invading DNA in vivo3,4. In contrast, pAgo from Aquifex aeolicus (AeAgo)6 and Natronobacterium gregoryi (NgAgo)7 use ssDNA guides to target RNA, suggesting a putative role in gene silencing, similar to that of eAgo, or in defense against RNA viruses. Moreover, other pAgos, like that of Rhodobacter spheroides (RsAgo), use RNA guides against DNA targets, maintaining its defense capability against invading DNA despite the absence of endonucleolytic activity in its PIWI domain8.\n\nHigh-resolution structures of pAgos in complex with guide and target DNAs support a mechanism of hydrolysis homologous to that of RNAse H, in which an Asp-Glu-Asp-Asp catalytic tetrad is formed at the cleavage site of its PIWI domain upon scanning and hybridization of gDNA and target ssDNA9. However, the actual mechanism for generation of the gDNA in vivo is essentially unknown and the described in vitro capability of the MjAgo5 and ThAgo10 apoproteins to cleave dsDNA (named DNA chopping) seems an unlikely mechanism for the generation of gDNA in vivo, as it cannot explain the observed inactivity against its own genomic DNA.\n\nFollowing description of the mechanism of action of ThAgo and PfuAgo, the possibility of using pAgos as tools for gene editing has been proposed, with the advantage of being easier to use than the CRISPR-Cas9 system11. However, attempts to directly use ThAgo for gene editing of mammalian cells were unsuccessful, likely due to the thermoactivity of this protein (unpublished results of our laboratory). Publication of gene editing of mammalian cells using NgAgo, a mesophilic pAgo from a hyperhalophilic archaea12, sparked controversy due to the inability of many other laboratories to reproduce the results13. Other published research has suggested that the substrates for NgAgo are RNA targets7. Despite this, the search has continued for new pAgos that could be successful in gene editing at low temperatures through a DNA-DNA interference mechanism11.\n\nRecently, an unreviewed preprint article describing the properties and structure of a mesophilic pAgo derived from Clostridium butyricum (CbAgo) has been posted online by the group of John van der Oost14. Here we show our independent work leading to the identification of a similar pAgo (CbcAgo) in the strain CWBI 1009 of C. butyricum, describing its properties in comparison to that of the CbAgo protein described in the preprint article, including a higher maximum temperature of activity, a strict requirement for 5’-phosphorylated gDNA and a smaller minimum gDNA size required for full activity.\n\n\nMethods\n\nThe search for mesophilic pAgos was performed using the web interface of the BLASTp program, with the protein sequence of Natronobacter georgii (WP_005580376.1) as a query, and directed to non-redundant GenBank CDS translations + PDB + SwissProt + PIR + PRF, excluding environmental samples from WGS projects. Using the default settings, proteins from two strains of Clostridium butyricum were identified (WP_045143632.1 and WP_058142162.1). Further BLASTp and COBALT analysis also with default settings revealed the presence of the four domains that characterize pAgos and the residues required for their likely nuclease activity within the PIWI domain. A fusion gene encoding an N-terminal Strep (II) tag and protein WP_045143632.1 from the strain C. butyricum CWBI1009 was synthesized (GenScript) following the codon usage of E. coli and cloned into a pET11d vector (Agilent Technologies) to generate the expression plasmid pET11-CbcAgo. For overexpression in E. coli KRX strain (genotype [F´, traD36, ΔompP, proA+B+, lacIq, Δ(lacZ)M15] ΔompT, endA1, recA1, gyrA96 (Nalr), thi-1, hsdR17(rk-, mk+), e14- (McrA-), relA1, supE44, Δ(lac-proAB), Δ(rhaBAD)::T7 RNA polymerase) (Promega), cultures were grown at 37 °C in LB with 100 μg/ml ampicillin until an optical density at 600 nm (OD600) of 0.7 was reached. CbcAgo expression was induced by the addition of 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) and 0.1% (w/v) of L-Rhamnose to the growth medium with further incubation at 20 °C for 18 h with mild shaking. This procedure resulted in the production of a protein of the expected size (90.4 kDa, 794 aa), as revealed by SDS-PAGE in comparison with size standards. After lysis by French press in TrisHCl 50mM, 1M NaCl, pH 8 buffer and elimination of insoluble debris by centrifugation (30 min / 30,000 × g / 4 °C) and filtration (Acrodisc Syringe Filter 0.45 μm, Life Sciences), the protein was purified by affinity chromatography in Strep-Tactin sepharose (IBA, Germany, Cat no. 2-1201-010). The CbcAgo protein concentration was measured by comparison with known concentration of BSA in Comassie-Blue stained SDS-PAGE using ImajeJ for quantification. Aliquots were stored at -20 °C with 40% glycerol until use. As a negative control for the interference assays, a double mutant lacking the catalytic tetrad (D541A, E577A) was generated (called DE mutant hereafter) by site-directed mutagenesis, (Quick change II site-directed mutagenesis kit, Agilent Technologies) overexpressed from plasmid pET11-CbcAgoDE and purified in the same way.\n\nProteins purified by this method were separated in an SDS-PAGE gel, digested with trypsin and chemotrypsin and the resulting peptides were identified by LC-MS/MS in an LTQ Orbitrap Velos Pro (high resolution, short gradient) equipment.\n\nThe synthetic gDNA and ssDNA targets described in Table 1 (SIGMA-ALDRICH) were used for the interference assays (Table 1). The standard interference assays were carried out as follows: after pre-incubation of the CbcAgo protein (6 μM) with a given primer (6 μM), selected among those described in Table 1, for 10 min at 37 °C in reaction buffer (20 mM Tris HCl, 150 mM NaCl, 2 mM of MnCl2, pH 7.5). The ssDNA target (1.2 μM) was added and the samples were incubated in the same buffer for 50 min at 37 °C, except when otherwise indicated. After incubation, the reaction was stopped by the addition to the sample of one reaction volume of loading buffer (85% formamide, 10 mM EDTA, 20% glycerol, 0.05% bromophenol blue, 0.05% xilencyanol) and further heating for 10 min at 100 °C. Separation of the ssDNA substrate, products and gDNA was carried out by electrophoresis in an 18–20% polyacrylamide gel in the presence of 6M urea (U-PAGE), using SYBR Gold Nucleic Acid Gel Stain for staining (Invitrogen S11494) and a UV spectrophotometer for detection. Synthetic oligonucleotides of different sizes were used as mobility standards.\n\nTable shows the names and sequence of the two target DNA used in this work (T-45 and T-50) and the oligonucleotides used as gDNA. All the gDNA except for 21-OH and 20-OH are phosphorylated at their 5’ end, being labelled as [phos].\n\nAssays of nicking activity on dsDNA were carried out following the above protocol using plasmid pMH184 isolated from E. coli cells in its supercoiled form as a target (GenJET Plasmid Miniprep Kit, Thermo scientific, Cat no. K0503). The molar ratio between CbcAgo: guide: dsDNA target was 3 : 6 : 0.0074 (μM). Reactions were stopped by adding 100 μg/mL of Proteinase K (Promega) and the products separated in agarose gels. As mobility standards, linear and nicked forms of pMH184 were generated by digestion with EcoRI (Thermo scientific, FD0275) and Nt.BspQI (Biolabs R06445) restriction enzymes, respectively.\n\n\nResults\n\nThe Strep II-tagged CbcAgo protein and its inactive DE derivative were overexpressed and purified by affinity chromatography (Figures 1A and 1B). In addition to the wild-type or DE mutant proteins, two smaller proteins were co-purified at low proportions even after repeated cycles of affinity chromatography. Mass spectrometry of proteolytic Trypsin/Chemotrypsin digestion fragments of these recalcitrant contaminant proteins revealed them to be the GroEL chaperone and an N-terminal fragment of the Strep II-tagged CbcAgo proteins (Figure 1C). Presence of these co-purified proteins did not interfere with the capacity of the wild-type CbcAgo protein to cleave the ssDNA target after being preloaded with a complementary gDNA guide (Figure 2). As the DE mutant was inactive in these assays, it was concluded that the endonuclease activity detected in the wild type was dependent on the presence of an active endonucleolytic site in CbcAgo’s C-terminal PIWI domain and was not as a result of hidden activity of the co-purified proteins.\n\nPurification by affinity chromatography of (A) wild-type or (B) inactive DE mutant. Lanes: M - molecular weight markers from top to bottom of 97.4, 66.2, 45 and 31 kDa; T - total cell protein fraction; S - soluble fraction; Sf - filtered soluble fraction; FT - flow through; W1-5 - fractions obtained upon addition of washing buffer; E1-5 - fractions obtained upon addition of elution buffer containing increasing concentrations of desthiobiotin. (C) Protein concentration of two independent preparations of wild-type CbcAgo and a single preparation of the DE mutant, compared with bovine serum albumin as standard (BSA). Protein identification was carried out by proteomic analysis.\n\nThe wild-type (Wt) and inactive DE mutant (DE) of the CbcAgo protein were pre-incubated with the indicated 5’ phosphorylated guide DNA for 10 min at 37 °C and further used to cut a complementary 45-nucleotide ssDNA target at the same temperature for 1 h. The reactions were carried out in the presence of 2 or 4 mM MnCl2; target (T), guide (G), and the major 34-mer product (P) of the reaction were identified in an 18% U-PAGE gel.\n\nOptimization of the DNA-DNA interference activity revealed a strict requirement for divalent cations, with higher activity shown in the presence of Mn2+ compared to Mg2+ (Figure 3), and a significant resistance to high ionic strength (Figure 4). Subsequent experiments were carried out with Mn2+ and 150 mM of NaCl in the interference buffer. Under these conditions, we then analyzed the range of temperatures at which this endonuclease was active, finding significant activity in the range of 25–55°C, with maximum activity between 37 and 42°C, as revealed by the absence of the target DNA band at this temperature, and no activity above 60°C (Figure 5). All subsequent experiments were carried out at 37°C.\n\nCbcAgo was loaded with a 21-mer, 5’-phosphorylated gDNA (G) and incubated with a complementary 45-mer ssDNA target (T) in reaction buffer in the absence (0) or presence of 2 or 4 mM Mn2+ or Mg2+. The major 34-mer product (P) of the reactions was identified in an 18% U-PAGE gel; target and guide were the same used in Figure 2.\n\nWild-type CbcAgo was preloaded with the same gDNA used in Figure 2 and incubated with the complementary 45-mer ssDNA target in the presence of the indicated concentrations of NaCl (M). Target (T), guide (G), and the major 34-mer product (P) of the reaction were identified in an 18% U-PAGE gel.\n\nWild-type CbcAgo was preincubated for 10 min at 37 °C with (+) or without (-) gDNA and then used in cleavage assays of an ssDNA target for 1 h at the indicated temperatures. The ssDNA target (T) and gDNA (G) were the same used in Figure 2.\n\nThe requirements of the gDNA were also analyzed, clearly showing a need for a 5’ phosphate end, as gDNAs with 5´-OH were unable to direct the enzyme to the complementary ssDNA target (Figure 6). The cutting site in the ssDNA target was also analyzed in detail using two gDNAs (20- and 21-mers) with a single nucleotide difference at the 5’-phosphorylated end, comparing the size of the largest product of the reaction with ssDNA size markers. As shown in Figure 7, the products obtained had sizes of 33 and 34 nucleotides for the 20- and 21-mer gDNA respectively. The localized cutting site in the target was complementary to the +10 and +11 position with respect to the 5´ end of the gDNA used.\n\nThe indicated 20- and 21-mer 5’phosphorylated (P) or unphosphorylated (OH) gDNAs were preincubated with CbcAgo and used in interference assays against the same complementary target. Presence (+) or absence (-) of CbcAgo or gDNA in the reaction are indicated. The target (T), guide (G) and the major 34-mer product (P) of the reaction were identified using an 18% U-PAGE gel.\n\nCbcAgo was loaded with 5’-phosphorylated 20- or 21-mer gDNA and used in interference cleavage assays against a 45-mer ssDNA target (T). The sizes of the products (P) were compared with ssDNA standards of the indicated sizes, using a 20% U-PAGE gel, leading to the conclusion that the cleavage site was complementary to the 10-11 base position of the gDNA.\n\nThe minimum size of the 5’P-guides was also studied. By shortening the gDNAs at their 3’ end and using them in interference assays against the same ssDNA target, we found similar efficiencies of cleavage up to a gDNA size of 11 nucleotides (Figure 8). Shorter gDNAs of 9- and even 7-mer still allowed the enzyme to partially cut the ssDNA target. Finally, the ability to direct the enzyme activity towards any desired site within a given ssDNA target was also demonstrated, using different guides of the same size but each with hybridization site displaced by a single base with respect to the next. Despite the fact that different efficiencies were detected, the capability of the guides to direct the wild-type CbcAgo activity against any designed site was clearly shown (Figure 9).\n\n(A) CbcAgo was incubated with 7-, 9-, 11-, 13-, 15-, 17- or 19-mer gDNAs complementary to a ssDNA target and used them in interference cleavage assays. The target (T), guide (G) and major product (P) of the reaction were identified using an 18% U-PAGE gel. (B) Sequences of the gDNAs and target ssDNA used in (A).\n\nCbcAgo was pre-loaded with a collection of 21-mer, 5´-phosphorylated gDNA that paired at positions displaced by a single nucleotide with a 45-mer ssDNA target (T). The products of the reactions were compared using a 20% U-PAGE gel with ssDNA markers of the indicated sizes (mer). (A) Assays with w1 to w4 gDNA. (B) Assays with gDNA w-5 to w-8. (C) Target DNA and gDNA used in A and B. Note that the cutting site was displaced along the target by a single nucleotide, always pairing at position 10-11 of the gDNAs (shaded triangle).\n\nFinally, assays on the activity of CbcAgo on dsDNA were carried out using supercoiled forms of plasmid pMH184 using four different guides complementary to the sense and antisense strands of the hygromycin resistance gene. All the gDNAs used were able to direct the production of a nick in the complementary strand, leading to the generation of open circle forms as the main product (Figure 10). However, using a combination of primers complementary to each strand did not result in the generation of linear forms of the plasmid (not shown).\n\n(A) CbcAgo was pre-loaded for 15 min at 37ºC with the Hygro-1 to 4 guides (lanes 1 to 4) and incubated for 16 hours at the same temperature with plasmid pMH184 that was essentially supercoiled (lane 5). Linear plasmid and nicked open circle were run in lanes 6 and 7 respectively. The molar ratio between CbcAgo:guide:target was 3 μM: 6 μM: 0.0074 μM. Reactions were stopped by adding 100 μg/mL of Proteinase K (Promega) and the products separated in agarose gels. (B) Sequence of the target in plasmid pMH184 paired with the gDNAs used in panel A.\n\n\nDiscussion\n\nNovel tools based on CRISPR-Cas9 RNA-DNA interference mechanisms have been developed in the last few years, taking a leading role among the current methods for gene editing. However, significant drawbacks must be overcome in order to allow their safe use in gene therapy, especially off-target actions and putative cellular persistence of the DNA used for the editing process. The description of pAgos in thermophilic bacteria and archaea that are able to target DNA using ssDNA guides has revealed the possibility of developing a new gene editing tool, in which a mix of the protein and its synthetic gDNA would be enough to produce specific cleavage without leaving anything behind that could produce deleterious long-term effects. This possibility was apparently supported by the publication of an article claiming the use of NgAgo to modify several genes in mammalian cell cultures12. However, the results were not reproducible by any of several groups13. For many microbiologists, the use of a protein from a hyperhalophilic archaea was suspect as they have evolved at the sequence level to tolerate very high potassium chloride concentrations as intracellular compatible solutes. Despite this, the article sparked a growing interest in finding an appropriate pAgo protein that could have this function, as thermophilic pAgos have little or no activity at mesophilic temperatures11.\n\nIn this context, the search for a mesophilic pAgo led our group (and that of J. van der Oost) to independently focus on a pAgo from Clostridium butyricum due to the mesophilic character of this organism and the presence of a pAgo in its genome that contains the catalytic tetrad at its PIWI domain. In a preprint article posted online14, the group of J. van der Oost provided an exhaustive description of the CbAgo protein but did not identify the exact strain they used. Although most of the findings of that article coincide with the properties of the CbcAgo protein described here, there are also some relevant differences that could relate either to differences in the sequence of the proteins or to the different methods used for the characterization.\n\nFirstly, in our study the CbcAgo protein seems slightly more stable, as we detected significant cleavage capacity of a ssDNA target at 55°C, whereas the preprint article described a 50°C limit for the activity of CbAgo. This could be related to differences in the protein sequence, as commented above, or to the presence of a chaperone (GroEL) in our samples that possibly protects the protein at 55°C. However, the main differences we found were concerning the requirement for 5´ phosphorylation of the gDNA (Figure 4) and the minimum gDNA size required for efficient cleavage (Figure 5). In our study, the requirement for phosphorylation of the gDNA was quite strict and no positive cutting was detected with any of the 5’-OH gDNAs used in different assays. This result is in agreement with the reported requirements of other pAgos such as ThAgo and PfAgo for gDNA phosphorylation3,4. Also, the minimum size for gDNA to be as efficient as longer gDNA in directing enzyme activity was 11 nucleotides, although we also detected some activity for gDNAs as short as 7 nucleotides (Figure 8). This contrasts with the minimum 12-mer required for binding of CbAgo to gDNA in single-molecule fluorescence assays and with the minimum 14-mer gDNA needed for cleavage of the ssDNA substrate reported for CbAgo14. The reasons for these discrepancies may be partly related to the method of detection used or the absence of phosphorylation in some of the gDNAs used in their work with the CbAgo protein14. Whatever the case, our data for minimum gDNA requirements is more similar to the 9 nucleotides described for TtAgo15.\n\nPutting our data in the context of the structure for CbAgo described in the figures of the preprint by the group of J. van der Oost14), and in that of ThAgo15 once even small gDNA are attached at a position of the MID domain defined by the gDNA 5’P-residue, CbcAgo is able to scan ssDNA for a matching sequence, approximating paired positions 10–11 to the active site of the PIWI domain, where the ssDNA target is cleaved in a cation-dependent manner. Smaller guides (i.e. 7-mers) may also function in the scanning, being only partially successful in guiding the substrate to the active site of the enzyme. This result supports the idea that CbcAgo needs a preloaded guide to fix its position on the target ssDNA and cut it efficiently, although the effectiveness of this cleavage seems to be related to the structure of the primer at the designated reaction temperature.\n\nAnother discrepancy has to do with the effects of CbcAgo on dsDNA. In this study, only nicking activity was detected when supercoiled plasmids were used as substrates (Figure 10). In fact, we did not detect the linearized plasmid form, even when two gDNAs (one for each strand) were used. However, in the preprint article14, plasmid linearization was detected at low yields, the activity being much more efficient in regions with low G+C content. This suggests that the CbcAgo protein alone is unable to open dsDNA after its nicking activity and that future directed evolution work will be needed for better adaptation to this type of substrate.\n\n\nData availability\n\nOpen Science Framework: Clostridium butyricum CWBI1009 Ago. https://doi.org/10.17605/OSF.IO/8GQUZ16\n\nThis project contains raw images of the gels used for each figure.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThis work was supported by a grant from the Spanish Ministry of Economy and Competitiveness [BIO2016-77031-R] to J. Berenguer. An institutional grant from Fundación Ramón Areces to the CBMSO is also acknowledged.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nTechnical assistance of E. Sánchez is acknowledged. The professional editing service NB Revisions was used for technical preparation of the text prior to submission.\n\n\nReferences\n\nMeister G: Argonaute proteins: functional insights and emerging roles. Nat Rev Genet. 2013; 14(7): 447–459. PubMed Abstract \| Publisher Full Text\n\nSwarts DC, Makarova K, Wang Y, et al.: The evolutionary journey of Argonaute proteins. Nat Struct Mol Biol. 2014; 21(9): 743–753. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwarts DC, Jore MM, Westra ER, et al.: DNA-guided DNA interference by a prokaryotic Argonaute. Nature. 2014; 507(7491): 258–261. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwarts DC, Hegge JW, Hinojo I, et al.: Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA. Nucleic Acids Res. 2015; 43(10): 5120–5129. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZander A, Willkomm S, Ofer S, et al.: Guide-independent DNA cleavage by archaeal Argonaute from Methanocaldococcus jannaschii. Nat Microbiol. 2017; 2: 17034. PubMed Abstract \| Publisher Full Text\n\nYuan YR, Pei Y, Ma JB, et al.: Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage. Mol Cell. 2005; 19(3): 405–419. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSunghyeok Y, Taegeun B, Kyoungmi, K, et al.: DNA-dependent RNA cleavage by the Natronobacterium gregoryi Argonaute. bioRxiv. 2017; 1–9. Publisher Full Text\n\nOlovnikov I, Chan K, Sachidanandam R, et al.: Bacterial argonaute samples the transcriptome to identify foreign DNA. Mol Cell. 2013; 51(5): 594–605. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSheng G, Zhao H, Wang J, et al.: Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage. Proc Natl Acad Sci U S A. 2014; 111(2): 652–657. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwarts DC, Szczepaniak M, Sheng G, et al.: Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute. Mol Cell. 2017; 65(6): 985–998 e986. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHegge JW, Swarts DC, van der Oost J: Prokaryotic Argonaute proteins: novel genome-editing tools? Nat Rev Microbiol. 2018; 16(1): 5–11. PubMed Abstract \| Publisher Full Text\n\nGao F, Shen XZ, Jiang F, et al.: DNA-guided genome editing using the Natronobacterium gregoryi Argonaute. Nat Biotechnol. 2016; 34(7): 768–773. PubMed Abstract \| Publisher Full Text\n\nLee SH, Turchiano G, Ata H, et al.: Failure to detect DNA-guided genome editing using Natronobacterium gregoryi Argonaute. Nat Biotechnol. 2016; 35(1): 17–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHegge JW, Swarts DC, Chandradoss SD, et al.: DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute. bioRxiv. 2019. Publisher Full Text\n\nWang Y, Sheng G, Juranek S, et al.: Structure of the guide-strand-containing argonaute silencing complex. Nature. 2008; 456(7219): 209–213. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerenguer J: Clostridium butyricum CWBI1009 Ago [Internet]. OSF. 2019. http://www.doi.org/10.17605/OSF.IO/8GQUZ" }	[ { "id": "51304", "date": "30 Jul 2019", "name": "Eduardo Santero", "expertise": [ "Reviewer Expertise Bacterial Molecular Biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGarcía-Quintans et al. reports on the identification of an Argonaute protein coding gene from the mesophilic bacterium Clostridium butyricum strain CWBI 1009, and characterise its product as a potential genome editing tool alternative to the well known CRISPR/Cas. Authors have purified the native protein and an inactive variant as a control, and thoroughly characterise its activity in relation to a number of parameters such as different temperatures, cations and ionic strength. Altogether, the work of García-Quintans is a well-designed characterisation of the in vitro activity of the CbcAgo protein.\nA similar work has been recently published in Nucleic Acids Research1, by the group of John van der Oost using a similar CbAgo protein from an unspecified strain of C. butyricum, as acknowledged by the authors. It appears that both proteins have very similar characteristics.\nThe authors claim some relevant differences between both proteins:\nEnzyme stability at different temperatures: In first place, none of authors assay thermostability, they assay activity at different temperatures, which is not the same. This should be changed in the Discussion, pg. 10. Based on the partial activity detected at 55°C in this paper, authors claim CbcAgo might be more “stable” than CbAgo (Discussion, pg. 10). However, CbAgo activity was not assayed at 55°C but at 50°C (partially active) and 64°C (inactive). A comparison of the activities at the same temperature, 50°C, which show almost maximal activity of CbcAgo but substantially reduced activity of CbAgo (<50%), would be more reliable. Anyways, since the difference is very small, it is very difficult to ascertain whether the differences are real or a consequence of slightly different assay conditions.\nStrict dependence on phosphorylation: Both CbAgo and CbcAgo, were unable to cut a short 45-mer target DNA if the gDNA is 5’-OH. However, Hegge et al., 20191, additionally reported partial activity of CbAgo on a longer target (120-mer), which was not tested in this manuscript. Therefore, in this regard, there is no data that supports the difference between both proteins claimed in this manuscript.\nMinimum size length of the gDNA: By comparing the results obtained with both proteins, it is apparent that CbcArgo requires a shorter gDNA to cleave the target. I think this is the most evident difference. However, as the authors acknowledge, these differences may be due to technical reasons rather than a difference in catalytic activity between the Argo proteins. The question of whether CbcArgo and CbArgo show any difference in activity could only be solved by making a side by side comparison of both proteins having the same tag, and using exactly the same procedure.\nAnyways, I really miss an alignment of CbArgo and CbcArgo proteins to know how different these proteins are at the amino acid level.\nOther comments: Please, properly align lanes and lanes names/numbers in Fig. 1. Requirement for 5´phosphorylated gDNA is shown in Fig. 6, not 4 (Discussion, pg. 10).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5126", "date": "09 Jan 2020", "name": "Jose Berenguer", "role": "Author Response", "response": "Reviewer 1. The study from Garcia-Quintans et al. looks at a new pAgo protein from Clostridium and investigates factors influencing its cleavage activity on DNA substrates. The in vitro activity is characterized pretty well, but there are some serious issues I find with the data and its presentation, as well as the contradictory findings of another recent publication. 1. The authors report a significant (by eye ~40%) contaminant of an N-terminal truncation at about half the size of the expected protein (Figure 1). I would assume this is an inactive form of the enzyme, but does it still bind guides? Bind to DNA targets? Perhaps affect the results of all the experiments in the paper? This should be addressed in more detail, and ideally cleaned up (along with the GroEL contaminat) using another chromatography step. Answer: As described in the article these contaminant proteins were systematically associated to the full length protein. The use of an inactive mutant cbAgo in which the N-terminal truncated fragment and the GroEL proteins were also co-purified guarantees that the observed activity on the target DNA in the presence of gDNA is exclusively due to the catalytic activity of the wt enzyme and not the result of hidden activity of the co-purified proteins. A series of different chromatographic steps have been tried, and in all cases separation of contaminants was not possible and/or the activity of the enzyme was diminished However we agree in that N-terminal fragment could bind either target or gDNA, leading to a minor subestimation of the actual activity of the enzyme or the optimal ratio target:gDNA, but without affecting the conclusions shown in the article. 2. Most of the gels are shown as zoomed in cropped sections of the gel. I feel these should instead show the whole, or at least more of, the gel, and include low-molecular weight marker standards. Some gels have oligonucleotide standards but the resolution is very poor in terms of distinguishing between a few bases (I'd suggest moving the guides by more than 1 base). And as shown in Figure 8 11 ntd ssDNA can clearly be seen, but where is it in the other gels where the product should be 2 ssDNA's? The most problematic is Figure 5 where the far right gel is too poor for publication, and seems to show production of P species without added guide at 55C? Where is the guide in all those wells? Figure 8 seems to have additional bands between P and guide, Figure 10 has an unidentified high molecular weight species, and the size markers in Figure 7 should be labeled more clearly. Answer: Due to requirements for organizing easy-to-follow figures, the images are cropped, as most figures in articles in the field are. However, as described in the article, the original full size images of all the gels used are accessible for detailed analysis at https://doi.org/10.17605/OSF.IO/8GQUZ Regarding gel resolution, we do not agree with the reviewer. Under the conditions used a single base makes a difference in the detected mobility of the gDNA and products, and our interest of using base-to –base changes was to be sure that we were able to move the cutting site in the tDNA in a precise manner (i.e. figs 7, 9). As there are so many size markers, only those closer to the size of the products were indicated, but likely the size of the numbers is too small for readers and are increased in the revised version. Regarding Fig 8, as the reviewer comments the reaction generates two ssDNA products from the tDNA. The largest one is 40 mer and is detected and labelled as P in the figures, and this is also the most adequate fragment for comparison. The small one is only 10 mer long (at the 3’ extreme of the tDNA) and escapes from the standard gels (lane 11 in Fig 8 shows the 11mer gDNA at the bottom of the gel). Also, in the other figures the small tDNA product moves out of the gels due to its small size. In figure 8, the reviewer points to the presence of minor bands below the main 40 mer product, especially under the lanes labelled 11 and 13. We cannot be sure of their origin but could correspond to lower affinity matches of the guides (11 and 13 mer) with the tDNA, as they are not detected with longer guides. Regarding figure 5, the reviewer is right. The line indicating absence (-) or presence (+) of guides have contracted during the figure assembly and it apparently seems that the enzyme cuts without guide at 50 and 55 ºC, but it is not that way. In the gel at right, the left lanes below each temperature (55, 60, 65 and 70 ºC) corresponds to assays without gDNA and the right one with gDNA (detected at the gel bottom), whereas in the left and central panels is first with (+) and then without (-) . In all cases, the product is only detected when the guide is present. This figure will be modified to correct the alignment of the labellings. Respect to Fig. 10, the high molecular size species detected could be concatenated plasmids, as it was detected in the untreated plasmid preparation (lane 5) and in the open circle treatment (lane 7) but not in the linear form (lane 6). 3. I feel there should be more explanation given to the (to me) bizarre finding that a 7 or 9 base guide can cut at the +10/11 position...which of course does not have a guided complement. How do the authors think this can happen? Answer: As the reviewer comments the ability of CbAgo to cut with low efficiency tDNA using guides so small that their 3’ end do not reach the catalytic residue in the protein where the cut is carried out was a surprising feature ( at least 11mer pairing would be needed as deduced from the structure in Hedge et al). Under our experimental conditions 5’-phosphorilated guides of 11 or more nucleotides are very efficient triggering the enzyme activity at complementary 10/11 position of the tDNA (Fig. 8). Smaller P-guides of 9 and even of 7 mer are still able to direct the enzyme to the expected cutting site, but with much lesser efficiency (Fig 8). The explanation that we find for this is that the small 5’-P- guides are also efficiently recognized by the MID domain of the protein and used to screen for complementarity. Once found (in a context without any other competing DNA) these small gDNAs could function as seed to position the tDNA near to the active site of the PIWI domain. Likely through thermodynamic movements, the target DNA eventually reach the catalytic domain in a sort of pendulum movement leading to the observed residual activity. This explanation has been included in the text for clarification 4. The authors mention the Hegge at al., preprint, which they should, but that paper was published in NAR after this study. And importantly, so was another study with CbAgo, from a strain mentioned here (Kuzmenko et al). In this study, the authors show several things at odds with the current work: no cleavage with 10 or 12-base guides even after 24hr incubation, activity to 60C, ability to use 5'-OH guides, the ability to cut dsDNA with opposite strand guides at 37C in 1-4h, and with moderate (500 nM) concentrations of CbAgo a chopping activity on plasmid DNA. It is likely this work was not available at the time the reviewed study was published, but it is difficult to ignore the contradictions now. It is possible that the Cb/CbcAgo protein is exactly the same in all 3 studies, and these discrepancies are significant for the conclusions presented here. Answer: As the reviewer indicates the article preprint by Kuzmenko was not available when the present article was sent for publication. The revised version includes the actual publication of both articles by Hegge et al and Kuzmenko et al in Nucleic Acids Research, both dealing with the same version of the CbAgo protein. The main discrepancies between our work and those mentioned above (which also show discrepancies between them) are the minimum size of the guides required for efficient cutting, smaller in our work, and the requirement for 5’-phosphorilation of our guides for efficient activity, and both are likely related. Phosphorilation of the guides: In the referred articles the use of 5’OH-gDNA under their experimental conditions allows the cutting of tDNA by CbAgo at lower efficiency than with phosphorylated guides (actually suggesting that the natural function for these proteins could require phosphorylated guides), whereas under our experimental conditions the CbcAgo protein was basically inactive at using 5’ OH-guides of 20 and 21 mer compared to the phosphorylated ones. The reasons underlying this difference could be related differnte and likely concurrent experimental differences. First, our gDNA hibridizes at the 3’ extreme of the tDNA instead of acting in the middle of the target as in Kuzmenko et al. Second, the size of the tDNA could also have an effect, as suggested by supplementary figure 4 of the article by Hegge et al in which a 5’OH guide does not cut a 45 mer target (identical in size to that used in our experiments), whereas it functions with a tDNA of 120 mer. Third, differences in the sequence of the CbAgo (used in Heddge and Kuzmenco works) and CbcAgo proteins (12 amino acid positions) or in their affinity taggings (Strep-tag in our work, His-tag in Kuzmenko´s and no tags in Heddge) could have effects on their performance. In any case, as commented above preference for 5’P guides for CbcAgo is clear in all the cases, and could be significant regarding its actual function in a cell context. Minimum size of the guides: The preference for 5’P guides by CbcAgo under our experimental conditions could also be the basis for experimental discrepancies regarding the minimum guide size to direct the protein, as in our experiments all our guides were phosphorilated, whereas in the works of Heddge et al the guides are labelled with a large fluorophore (Cy3), and in that of Kuzmenko et al. the guides hybridize in the middle of larger tDNA instead of pairing starting at the 3’ extreme of the tDNAs as we did in our experimental setting. DNA chopping: Another point raised by the reviewer focus on the so called “chopping” activity of the enzyme on double stranded DNA. In our hands, we did not observe any degradative activity on the plasmid used as target (pMH184) even after 16 hours of incubation at 37ºC with significant concentrations of CbAgo protein loaded with gDNA (Fig 10) or without gDNA (experiment not shown). dsDNA activity: Regarding cutting of supercoiled plasmid, it seems clear that nicks were generated using CbcAgo loaded with guides directed to both DNA strands of a supercoiled plasmid, but under our experimental condition we did not detect linearized plasmid, supporting interference between overlapping cutting sites 5. Related, I'd expect there to be some plasmid chopping given the time and concentrations the authors describe. But no Apo reactions are shown in Figure 10, an important control that is left out. And a comparison of attempts to digest non-supercoiled plasmid would be good for the explanation that dsDNA cannot be accessed w/o supercoiling. Answer: As commented above, no chopping was detected in our experiments despite incubation for 16 h at 37ºC with the protein in nmolar ratios 10:1 with the dsDNA plasmid 6. Minor points, but there are some errors (\"xilencyanol\", \"ImajeJ) and inconsistencies (PfAgo/PfuAgo) that should be fixed. Answer: Corrected in the revised form" } ] }, { "id": "51940", "date": "02 Aug 2019", "name": "Nathan A. Tanner", "expertise": [ "Reviewer Expertise DNA enzymes and polymerases", "biochemistry and molecular biology", "biotechnology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study from Garcia-Quintans et al. looks at a new pAgo protein from Clostridium and investigates factors influencing its cleavage activity on DNA substrates. The in vitro activity is characterized pretty well, but there are some serious issues I find with the data and its presentation, as well as the contradictory findings of another recent publication.\nThe authors report a significant (by eye ~40%) contaminant of an N-terminal truncation at about half the size of the expected protein (Figure 1). I would assume this is an inactive form of the enzyme, but does it still bind guides? Bind to DNA targets? Perhaps affect the results of all the experiments in the paper? This should be addressed in more detail, and ideally cleaned up (along with the GroEL contaminat) using another chromatography step.\n\nMost of the gels are shown as zoomed in cropped sections of the gel. I feel these should instead show the whole, or at least more of, the gel, and include low-molecular weight marker standards. Some gels have oligonucleotide standards but the resolution is very poor in terms of distinguishing between a few bases (I'd suggest moving the guides by more than 1 base). And as shown in Figure 8 11 ntd ssDNA can clearly be seen, but where is it in the other gels where the product should be 2 ssDNA's? The most problematic is Figure 5 where the far right gel is too poor for publication, and seems to show production of P species without added guide at 55C? Where is the guide in all those wells? Figure 8 seems to have additional bands between P and guide, Figure 10 has an unidentified high molecular weight species, and the size markers in Figure 7 should be labeled more clearly.\n\nI feel there should be more explanation given to the (to me) bizarre finding that a 7 or 9 base guide can cut at the +10/11 position...which of course does not have a guided complement. How do the authors think this can happen?\n\nThe authors mention the Hegge et al., preprint, which they should, but that paper was published in NAR after this study. And importantly, so was another study with CbAgo, from a strain mentioned here (Kuzmenko et al.1). In this study, the authors show several things at odds with the current work: no cleavage with 10 or 12-base guides even after 24hr incubation, activity to 60C, ability to use 5'-OH guides, the ability to cut dsDNA with opposite strand guides at 37C in 1-4h, and with moderate (500 nM) concentrations of CbAgo a chopping activity on plasmid DNA. It is likely this work was not available at the time the reviewed study was published, but it is difficult to ignore the contradictions now. It is possible that the Cb/CbcAgo protein is exactly the same in all 3 studies, and these discrepancies are significant for the conclusions presented here.\n\nRelated, I'd expect there to be some plasmid chopping given the time and concentrations the authors describe. But no Apo reactions are shown in Figure 10, an important control that is left out. And a comparison of attempts to digest non-supercoiled plasmid would be good for the explanation that dsDNA cannot be accessed w/o supercoiling.\n\nMinor points, but there are some errors (\"xilencyanol\", \"ImajeJ) and inconsistencies (PfAgo/PfuAgo) that should be fixed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5127", "date": "09 Jan 2020", "name": "Jose Berenguer", "role": "Author Response", "response": "Reviewer 2 García-Quintans et al. reports on the identification of an Argonaute protein coding gene from the mesophilic bacterium Clostridium butyricum strain CWBI 1009, and characterise its product as a potential genome editing tool alternative to the well-known CRISPR/Cas. Authors have purified the native protein and an inactive variant as a control, and thoroughly characterise its activity in relation to a number of parameters such as different temperatures, cations and ionic strength. Altogether, the work of García-Quintans is a well-designed characterisation of the in vitro activity of the CbcAgo protein. A similar work has been recently published in Nucleic Acids Research1, by the group of John van der Oost using a similar CbAgo protein from an unspecified strain of C. butyricum, as acknowledged by the authors. It appears that both proteins have very similar characteristics. The authors claim some relevant differences between both proteins: Enzyme stability at different temperatures: In first place, none of authors assay thermostability, they assay activity at different temperatures, which is not the same. This should be changed in the Discussion, pg. 10. Based on the partial activity detected at 55°C in this paper, authors claim CbcAgo might be more “stable” than CbAgo (Discussion, pg. 10). However, CbAgo activity was not assayed at 55°C but at 50°C (partially active) and 64°C (inactive). A comparison of the activities at the same temperature, 50°C, which show almost maximal activity of CbcAgo but substantially reduced activity of CbAgo (<50%), would be more reliable. Anyways, since the difference is very small, it is very difficult to ascertain whether the differences are real or a consequence of slightly different assay conditions. Answer: In a further article by Kuzmenko et all, the CbAgo protein shows nuclease activity up to 60ºC, so the apparent thermostability differences are more likely due to the experimental settings used than to the protein sequence itself. We have modified the discussion accordingly just saying that the apparent activity at high temperature is higher under our experimental conditions, but that due to differences in the assays (dDNA and tDNA) the data cannot be directly compared Strict dependence on phosphorylation: Both CbAgo and CbcAgo, were unable to cut a short 45-mer target DNA if the gDNA is 5’-OH. However, Hegge et al., 20191, additionally reported partial activity of CbAgo on a longer target (120-mer), which was not tested in this manuscript. Therefore, in this regard, there is no data that supports the difference between both proteins claimed in this manuscript. Answer: This has been already commented as answer to point 4 raised by reviewer 1. We agree in that even for the CbAgo it is more likely to use a 5’P gDNA than an 5’-OH one, as i) they are less active in the best of the cases than 5’-P counterparts, and ii) the binding site at the MID domain shows several contacts between the 5’P extreme of the gDNA and residues in the protein, supporting a higher affinity for this type of substrates than for 5’OH ones. The differences found are more likely due to differences in the experimental setting, including the selection of the substrates and the hybridization position. Minimum size length of the gDNA: By comparing the results obtained with both proteins, it is apparent that CbcArgo requires a shorter gDNA to cleave the target. I think this is the most evident difference. However, as the authors acknowledge, these differences may be due to technical reasons rather than a difference in catalytic activity between the Argo proteins. The question of whether CbcArgo and CbArgo show any difference in activity could only be solved by making a side by side comparison of both proteins having the same tag, and using exactly the same procedure. Anyways, I really miss an alignment of CbAgo and CbcAgo proteins to know how different these proteins are at the amino acid level. Answer: Proteins CbAgo (used in the articles of Hedgge et al and Kuzmenko et al and CbcAgo (Used in this article) are very similar. From the 748 amino acids of the three protein only 12 differences exist: N188D, D191G, R200K, S204A, E212K, K216N, S217T, E220D, K253N, K258Q, I343V, S466L, being the first position corresponding to the CbAgo and the second to the CbcAgo. These changes are located mainly at inter-domain regions and could be not too relevant for the activity but could affect parameters such as stability. Additional differences in the results between both proteins could be due to the presence or absence of tags (Strep-tag in CbcAgo, His-tag in Kuzmenko´s CbAgo and no tags in Heddge`s CbAgo). Other comments: Please, properly align lanes and lanes names/numbers in Fig. 1. Answer: OK, done Requirement for 5´phosphorylated gDNA is shown in Fig. 6, not 4 (Discussion, pg. 10). Answer: OK, Modified Answer: Actualized references included in the text: Hegge, J. W. et al. DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute. Nucleic Acids Res. 47, 5809-5821. doi: 10.1093/nar/gkz306.(2019). Kuzmenko, A., Yudin, D., Ryazansky, Kulbachinskiy, S. A. & Aravin, A. A. Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rose. Nucleic Acids Res. 47, 5822–5836 doi: 10.1093/nar/gkz379 (2019)" } ] } ]	1	https://f1000research.com/articles/8-321
https://f1000research.com/articles/7-146/v1	05 Feb 18	{ "type": "Systematic Review", "title": "Epidemiological evidence relating environmental smoke to COPD in lifelong non-smokers: a systematic review", "authors": [ "Peter N. Lee", "Barbara A. Forey", "Katharine J. Coombs", "Jan S. Hamling", "Alison J. Thornton", "Barbara A. Forey", "Katharine J. Coombs", "Jan S. Hamling", "Alison J. Thornton" ], "abstract": "Background: Some evidence suggests environmental tobacco smoke (ETS) might cause chronic obstructive pulmonary disease (COPD). We reviewed available epidemiological data in never smokers. Methods: We identified epidemiological studies providing estimates of relative risk (RR) with 95% confidence interval (CI) for various ETS exposure indices. Confounder-adjusted RRs for COPD were extracted, or derived using standard methods. Meta-analyses were conducted for each exposure index, with tests for heterogeneity and publication bias. For the main index (spouse ever smoked or nearest equivalent), analyses investigated variation in RR by location, publication period, study type, sex, diagnosis, study size, confounder adjustment, never smoker definition, and exposure index definition. Results: Twenty-eight relevant studies were identified; nine European or Middle Eastern, nine Asian, eight American and two from multiple countries. Five were prospective, seven case-control and 16 cross-sectional. The COPD definition involved death or hospitalisation in seven studies, GOLD stage 1+ criteria in twelve, and other definitions in nine. For the main index, random-effects meta-analysis of 33 heterogeneous (p<0.001) estimates gave a RR of 1.20 (95%CI 1.08-1.34). Higher estimates for females (1.59,1.16-2.19, n=11) than males (1.29,0.94-1.76, n=7) or sexes combined (1.10,0.99-1.22, n=15 where sex-specific not available), and lower estimates for studies of 150+ cases (1.08,0.97-1.20, n=13) partly explained the heterogeneity. Estimates were higher for Asian studies (1.34,1.08-1.67, n=10), case-control studies (1.55,1.04-2.32, n=8), and COPD mortality or hospitalisation (1.40,1.12-1.74, n=11). Some increase was seen for severer COPD (1.29,1.10-1.52, n=7). Dose-response evidence was heterogeneous. Evidence for childhood (0.88,0.72-1.07, n=2) and workplace (1.12,0.77-1.64, n=4) exposure was limited, but an increase was seen for overall adulthood exposure (1.20,1.03-1.39, n=17). We discuss study weaknesses that may bias estimation of the association of COPD with ETS. Conclusions: Although the evidence suggests ETS increases COPD, study weaknesses and absence of well-designed large studies precludes reliable inference of causality. More definitive evidence is required.", "keywords": [ "Passive smoking", "COPD", "Dose-response", "Meta-Analysis", "Review", "Pulmonary Disease" ], "content": "Introduction\n\nThis systematic review aims to present an up-to-date meta-analysis of available epidemiological evidence relating exposure to environmental tobacco smoke (ETS) to risk of chronic obstructive pulmonary disease (COPD) in lifelong non-smokers (“never smokers”). As described below, this review considers data from 28 longitudinal, case-control or cross-sectional studies1–28.\n\nIt is long established that active smoking causes COPD, the U.S. Surgeon General concluding in 196429 that “cigarette smoking is the most important of the causes of chronic bronchitis in the United States, and increases the risk of dying from chronic bronchitis”. This opinion was echoed in their 2004 report30, which felt the evidence “sufficient to infer a causal relationship between active smoking and chronic obstructive pulmonary disease morbidity and mortality”, a view confirmed by a recent systematic review31.\n\nSidestream smoke (released between puffs from the burning cone) contains similar chemicals to mainstream smoke (drawn and inhaled by smokers), but with different relative and absolute quantities of many individual constituents32. However, sidestream smoke, after mixing with aged exhaled mainstream smoke, is diluted massively by room air before non-smokers inhale it. Smoke constituent levels in tissues of non-smokers are very much lower than in smokers, studies using cotinine typically indicating a relative exposure factor between 0.06% and 0.4%33–35, with studies using particulate matter indicating a lower factor of 0.005% to 0.02%36–44. Though an effect of ETS on COPD risk is plausible, it is difficult to establish this with certainty, as a threshold is a logical possibility. The same difficulty of establishing effects of ETS exposure on other diseases caused by smoking is also present, notably for lung cancer31,45.\n\nIn 2006, a review by the U.S. Surgeon General of the association of COPD with ETS exposure46 concluded that “the evidence is suggestive but not sufficient to infer a causal relationship between second-hand smoke exposure and risk for COPD”, the need for additional research also being highlighted. Although that review cited only nine of the 28 studies considered here1,3,5–10,13, and although various new studies have appeared since then, no other fully comprehensive review of this subject appears to have been undertaken.\n\nThis review, which is essentially an update of the 2006 review46 is an attempt to assess the epidemiological evidence currently available, restricting attention to studies of COPD in which its relationship to one or more ETS exposure indices has been studied in never smokers. This restriction to never smokers is necessary as there is a very strong association of COPD with smoking46, and it is difficult to reliably detect any ETS effect where a history of smoking is present. This is because the extent of a smoker’s overall exposure to smoke constituents is determined largely by his own smoking habits and hardly at all by his much smaller ETS exposure, and also because smoking and ETS exposure are correlated (e.g. since smokers tend to marry smokers). Any errors in assessing smoking history are therefore likely to cause a residual confounding effect much larger than any plausible ETS effect47.\n\nAs the 2006 US Surgeon General’s Report46 notes “COPD is a non-specific term, defined differently by clinicians, pathologists, and epidemiologists, each using different criteria based on symptoms, physiologic impairment, and pathologic abnormalities”. That report goes on to state that “the hallmark of COPD is the slowing of expiratory airflow measured by spirometric testing, with a persistently low FEV1 [forced expiratory volume in one second] and a low ratio of FEV1 to FVC [forced vital capacity] despite treatment”. International guidelines48 define COPD as post-bronchodilator FEV1/FVC <0.70, with severity classified by subdividing FEV1 as a percentage of predicted into four groups (≥80, <80, <50 and <30%). The term COPD was little used until the 1980s, and diagnoses commonly used earlier (e.g. chronic bronchitis and emphysema) do not correspond exactly to what is now termed COPD. The studies we selected for review used disease definitions close enough to COPD as now defined to reasonably allow overall assessment. Some studies present additional results using criteria corresponding to severer forms of the disease. While these data are presented here, they are not included in our detailed meta-analyses.\n\n\nMaterials and methods\n\nThis systematic review was conducted according to PRISMA guidelines49.\n\nAttention is restricted to epidemiological longitudinal, case-control or cross-sectional studies which provide risk estimates for never (or virtually never) smokers for any of the following indices of ETS exposure: spouse, partner, cohabitant, at home, at work, in adulthood, in childhood.\n\nThe term COPD is relatively recent, so we also included studies with outcomes described otherwise. Following the strategy used in our review of smoking and COPD31, outcomes “could be based on International Classification of Diseases (ICD) codes, on lung function criteria, on a combination of lung function criteria and symptoms, or on combinations of diagnosed conditions….where diagnoses were extracted from medical records or reported in questionnaires”. Acceptable combinations of diagnosed conditions had to include both chronic bronchitis and emphysema, but could also additionally include asthma, acute and unqualified bronchitis or bronchiectasis. However, studies were rejected where results were only available for emphysema, for chronic bronchitis, for respiratory symptoms such as cough or phlegm, or for lung function criteria not equating to COPD. Over-broad definitions such as respiratory disease were also not accepted. Acceptable lung function criteria included those of the Global Initiative for Chronic Obstructive Lung Disease (GOLD), the European Respiratory Society, and the British and American Thoracic Societies,\n\nStudies which provide near equivalent definitions of “never smokers” are also accepted; thus never smokers can include occasional smokers or smokers with a minimal lifetime duration of smoking or number smoked. Risk estimates may be based on relative risks (RRs), hazard ratios (HRs), or odds ratios (ORs), and must either be provided directly or be capable of being estimated from the data provided.\n\nA PubMed search identified papers published up to June 2016 using the term “COPD AND (ENVIRONMENTAL TOBACCO SMOKE OR PASSIVE SMOKING OR SECONDHAND SMOKE EXPOSURE OR INVOLUNTARY SMOKING)”, with restriction to humans. After rejecting papers that were clearly irrelevant based on the abstract, copies of the others were obtained for inspection. Other potentially relevant papers were obtained from reference lists in the 2006 Surgeon General report46, an earlier review we conducted47 and relevant review papers identified in the search. The complete list of potentially relevant papers were then looked at in detail to determine those which described studies satisfying the selection criteria, the rejected papers also including those where an alternative paper provided results from the same study that were more useful (e.g. based on a longer follow-up, a larger number of cases, or using a disease definition closer to COPD as currently defined).\n\nDetails were extracted from relevant publication on the following: study author; year of publication; study location; study design; sexes included; disease definition; number of cases; potential confounding variables considered; and never smoker definition. An effect estimate together with its associated 95% confidence interval (CI) was obtained, where available, for ETS exposure at home, at work, in adulthood, childhood, and from these sources combined. Choice between multiple definitions of COPD followed the rules of Forey et al.31, except that here we also obtained additional estimates, if available, for severer COPD. We preferred effect estimates where the denominator was with no (or minimal) exposure to the ETS type considered rather than with no exposure to any ETS. Effect estimates and 95% CIs extracted were sex-specific, if possible, and for longitudinal studies were for the longest follow-up available. Estimates adjusted for covariates, where available, were generally preferred to unadjusted estimates, except that results adjusted for symptoms or precursors of COPD were not considered. Where a study provided multiple adjusted estimates, we used that adjusted for most covariates. Dose-response data were also extracted, where available.\n\nFor a study reporting effect estimates and CIs only by exposure level, that for the overall unexposed/exposed comparison was estimated using the Morris and Gardner method50 for unadjusted data or the Hamling et al. method51 for adjusted data. These methods also allowed estimation of the significance of dose-related trends, if not given in the source publication.\n\nAs the great majority of effect estimates were ORs derived from case-control or cross-sectional studies, and as the RRs or HRs from longitudinal studies were all based on low incidences, where the OR would be virtually the same, all estimates were treated as if they were ORs. In the rest of this paper, we use OR rather than referring to specific types of effect estimate.\n\nA pre-planned set of fixed-effect and random-effects meta-analyses were carried out using standard methods52. Heterogeneity was quantified by H, the ratio of the heterogeneity chi squared to its degrees of freedom. The I-squared statistic53 is related to H by the formula I2 = 100 (H-1)/H. Publication bias tests were also conducted using the Egger method54.\n\nOur main analyses included OR estimates for the exposure most closely equivalent to “spouse ever smoked” where results were provided or could be estimated. This selection was based on the source of exposure (spouse highest preference, then partner, cohabitant, home or work). Spousal smoking is traditionally used for studying possible ETS effects, it being clearly demonstrated that women married to a smoker have much higher cotinine levels than women married to a non-smoker55. Apart from the meta-analyses using all available estimates, meta-analyses also investigated variation in the OR according to a list of pre-defined factors, and using the following subsets: continent (North America, Asia, Europe, multicountry); publication period (1976–1990, 1991–2005, 2006–2016); study type (longitudinal, case-control, cross-sectional); sex (males, females, combined); diagnosis (mortality or hospitalisation, GOLD stage 1+, other); method of taking asthma into account (included as part of the COPD definition, adjusted for, asthmatic participants excluded, ignored); number of cases estimate based on (<50, 50–149, 150+ cases); extent of confounder adjustment (unadjusted for age, adjusted for age and at most four other variables, adjusted for age and five or more variables); never smoker definition (never smoked any product, never smoked but product unstated, other – including never cigarette smoker, occasional smoker or very short-term smoker); and definition of exposure index (spouse specifically, other exposure at home, other).\n\nMeta-analyses were also carried out for the main index using the estimates for severer COPD, and also for other indices of exposure with sufficient data (workplace, overall adult – including at least home and work, childhood). Here, data were too limited to study variation in the OR by the subsets described above.\n\nResults of the overall meta-analyses are displayed as forest plots. In each plot, individual estimates are listed in increasing order of the OR. For the main index, estimates are grouped by region. Random-effects estimates are also shown. The estimates are not only shown numerically, but in graphical form on a logarithmic scale, where the OR is shown as a square, the area of which is proportional to its inverse-variance weight. Arrows warn when the CI goes outside the range of the plot.\n\nWe did not attempt to derive any overall score based on study quality and risk of bias for each individual study, as the relative importance of different sources of bias or poor study quality is difficult or impossible to assess accurately. Instead, we attempted to gain insight into this in two ways. First, as mentioned in the previous section, we carried out meta-analyses showing how the OR varied by some relevant aspects linked to study quality and bias, such as study size, study type, source of diagnosis, method of taking asthma into account, and extent of confounder adjustment. Second, we considered factors affecting quality and bias in the discussion section, including some factors that affected all or virtually all of the studies.\n\n\nResults\n\nThe PubMed search produced 509 hits. As summarized in Figure 1, Seventy-five were considered of potential relevance based on the abstracts, 15 of which proved to meet the inclusion criteria on examination of the papers themselves. Further examination of reference lists in reviews46,47,56–63 and in papers obtained identified a further 40 papers of potential relevance, 13 of which met the inclusion criteria. Of the 87 papers examined but not accepted, the most common reasons for rejection were no results for never smokers (38 papers), not COPD as defined (26), no control group or no results for unexposed participants (11) and better results for the same cohort given in another paper (9), some studies being rejected for more than one reason. Supplementary File 1 gives details of the studies rejected and fuller reasons for rejection.\n\nThe flow-chart shows the number of articles identified from the PubMed search and from reference lists of reviews and articles obtained, as well as showing those excluded, with reasons for exclusion. Note that some articles were excluded for multiple reasons.\n\nTable 1 gives details of the 28 epidemiological studies that met the inclusion criteria, including author, reference(s), publication year, location, design, sexes included, disease definition, account taken of asthma, and numbers of cases in never smokers. The studies are listed in chronological order of publication and are given consecutive identifying study numbers.\n\na First author and reference of principal publication\n\nb Year of publication\n\nc Study types are CC = case-control, CS = cross-sectional, L = longitudinal. For longitudinal studies, number of years follow-up is shown\n\nd Age at baseline for longitudinal studies\n\ne Definition of principal COPD outcome is shown. Definition of severer COPD used is shown in footnotes, along with alternative definitions for which results are available\n\nf Number of cases in lifelong non-smokers\n\ng Study also included females, but none had COPD\n\nh No mention of use of bronchodilator prior to spirometry\n\ni Named as chronic bronchitis, but defined by authors as ICD 9th revision 491, 492, 496 so equates to COPD\n\nj Ignored for controls\n\nk Analysed as a nested CC study\n\nl Never smoking women had been identified by earlier studies in the same areas\n\nm Approximate estimate\n\nn Severer outcome definition based on GOLD Stage 2+\n\no Alternative results are also available for GOLD stage 0+\n\np Severer outcome definition based on NICE criteria (FEV1/FVC <0.7 and FEV1 <80% predicted) described as equivalent to GOLD stage 2+ (no bronchodilator, omitting participants with diagnosis of asthma). Alternative results are also available based on the LLN criteria, for “clinically significant COPD” based on the LLN, GOLD and NICE criteria, and including participants with diagnosis of asthma\n\nq Alternative results are also available using the LLN criteria\n\nr Based on death certificate, supplemented by medical records for lung function if their death was not from COPD\n\ns Severer outcome definition is based on LLN criteria plus FEV1<80% predicted\n\nThe included studies are mainly of representative populations, except that studies 18 and 26 have a large proportion with respiratory symptoms. Of the 28 studies, one was published in the 1970s, six in the 1980s, one in the 1990s, nine between 2000 and 2009 and 11 more recently.\n\nNine studies were conducted in Europe or the Middle East, subsequently referred to as “Europe” (two in England, and one each in Greece, Italy, Lebanon, Poland, Sweden, Switzerland and Turkey), while nine took place in Asia (five in China, and one each in Hong Kong, Japan, Korea and Taiwan), seven in North America (six in the USA and one in Canada) and one in South America (Brazil). Two studies presented combined results, one from 16 countries, the other from 14.\n\nFive studies were longitudinal in design, with the length of follow-up varying from 12 to 39 years, one was a cross-sectional study analysed as a nested case-control study, 16 other studies were cross-sectional, with the remaining six of case-control design.\n\nMost studies were of both sexes, though six studies considered only females.\n\nTwo studies considered those with a minimum age of 60, with a further 16 having a minimum age between 35 and 51. Other studies had a lower minimum age.\n\nDefinitions of outcome used varied by study. Seven studies required the case to have died or been hospitalised for COPD, while a further 12, mainly relatively recent cross-sectional studies, used COPD as defined by the GOLD stage 1+ criteria. The remaining nine studies used other definitions, as detailed in Table 1. Five studies (17, 19, 20, 26, 28) also provided results for severer COPD (generally equivalent to GOLD 2+, see footnotes to Table 1).\n\nTwenty-one studies ignored asthma in their outcome definition and analysis, with the remaining 12 studies equally divided into those that included asthma in their outcome definition, excluded asthmatics, or adjusted for asthma status in analysis.\n\nMost studies were small, with ten studies considering less than 100 cases and only one study (15) more than 1000 cases.\n\nTable 2 gives the adjustment variables used and the definitions of never smokers used in the studies.\n\na Never any product = never smoked cigarettes, pipes or cigars; Never NOS = never smoked, product unspecified;\n\nb Results adjusted for more variables not used as adjustment included health status\n\nc Questions on pipe and cigar smoking were asked at baseline, but not at the follow-up interviews\n\nd The cases and controls were matched on age\n\nFive studies (1, 20, 21, 24, 25) made no adjustment for any potential confounding variables, while some others made little or no adjustment for such variables as occupation, education, diet and family history of disease, which may differ between smoking and non-smoking households64. Failure to adjust for household size, where the index of exposure is based on presence of a smoker in the household, was also common. Where adjustment was carried out, all but four studies considered age, although study 3 adjusted for the husband’s age rather than the subject’s.\n\nFifteen studies were of never smokers, though only three of these made it clear they were never smokers of cigarettes, pipes or cigars. Five studies were of never cigarette smokers (i.e. they may have included some pipe or cigar only smokers), the remaining eight allowing a minimal smoking history, such as smoking less than 1 cigarette a day or less than 100 cigarettes in life.\n\nThe main meta-analyses use an exposure index that relates as closely as possible to ever smoking by the spouse. Table 3 shows the definitions of ETS exposure used for the main index. This was based on smoking by the spouse for five studies, and on smoking by cohabitants for a further 13 (although study 13 only included participants who had lived with a smoker 10 years previously, and study 20 only considered ETS exposure in the home in the two weeks prior to the study). For the remaining studies, the index was based on exposure in the home and at work (studies 4, 12, 17, 18 and 27) or on a combination of exposure from any source (studies 11, 15, 19, 21 and 25).\n\nAlthough most studies presented results comparing participants who were exposed or unexposed to ETS, some required a minimum level before a subject could be classified as exposed. In study 19, exposure had to be for at least one hour per week, while study 12 specified living with a smoker who smoked in the home or exposure at work for at least one hour per day. In studies 20 and 28, exposure had to have been in the previous two weeks, while participants in study 25 had to have had regular exposure in the previous year. In study 22 exposure had to be for 15+ minutes per day at least once per week for two or more years, while in study 15 the minimum requirement was 15 minutes or more, three or more times per week. In study 11, participants were only considered to have been exposed if they reported four or more hours of exposure on most days or nights in the previous year. Finally, study 14 required 10 years of exposure.\n\nTable 3, supported by Figure 2, also presents the ORs for the main exposure index, while Table 4 presents the results of meta-analyses, and Table 5 the dose-response data.\n\nIndividual study estimates of the OR and its 95%CI are shown separately by region, sorted in increasing order of OR. These are shown as numbers, and also graphically on a logarithmic scale. Random-effects estimates of ORs and 95%CIs are also shown for each region combined and overall. Studies are identified by the study number shown in Table 1. In the graphical representation, ORs are indicated by a square, with the area of the square proportional to the weight.\n\nFrom Table 3 it can be seen that, of the 33 individual OR estimates given for COPD, 24 are above 1.00, seven of these increases being significant at p<0.05. Eight studies reported an OR below 1.00, but only in study 4 for females was the reduction statistically significant. Study 25 reported an OR of 1.00, while study 24, excluded from the meta-analyses, did not present an OR but reported no significant relationship with duration or type of exposure. In addition, five studies presented a total of seven OR estimates for severer COPD, with five estimates above 1.00 (one significantly so and one marginally significant) and two non-significantly below 1.00.\n\na Or nearest equivalent to spouse or household member (see text and Table)\n\nb Study types are CC = case-control, CS = cross-sectional, L = longitudinal. For longitudinal studies, number of years follow-up is shown\n\nc Comparison is with those not exposed as defined, except where indicated otherwise\n\nd RRs from longitudinal studies are taken as being equivalent to ORs\n\ne Separate results also available for current smoker and exsmoker\n\nf OR and/or CI estimated from data provided\n\ng Approximate estimates\n\nh Compares exposed at home only to unexposed. Excludes those exposed at work\n\ni A straw cigarette is a handful of tobacco, wrapped in a corn husk; study not included in meta-analysis\n\nj Compared to subjects not exposed to any source of ETS; results also available for current or former exposure\n\nk Results not included in the meta-analysis in figure 2\n\nTable 4 demonstrates that the overall evidence for the main exposure index shows some increased risk of COPD, with the random-effects OR, based on 33 independent estimates, being 1.20 (95% CI 1.08-1.34) with no clear evidence of publication bias (0.05<p<0.1), but clear heterogeneity (p<0.001). The largest contributors to this were the high ORs for studies 18 and 21 and in females for study 13 and the low OR in females for study 4.\n\na Definition of COPD as shown in the body of Table 1, severer COPD in footnotes to Table 1. Data as shown in Table 3\n\nb Or nearest equivalent to spouse or household member (see text and Table 3)\n\nc Heterogeneity relates to variation between studies within subgroup, except for results given in italics which relate to heterogeneity between subgroups\n\nd N number of estimates in meta-analysis\n\ne Egger test p expressed as <0.001, <0.01, <0.05, <0.1 or NS (p≥0.1)\n\nf DF degrees of freedom\n\ng p expressed as <0.001, <0.01, <0.05, <0.1 or NS (p≥0.1)\n\nh Includes one study from Turkey and one from Lebanon\n\ni Including study 22\n\nAlthough there was no significant heterogeneity by continent, a significant increase was seen for North America (1.19, 1.01-1.41, n = 10) and Asia (1.34, 1.08-1.67, n = 10), but not for other locations. There was no significant heterogeneity by period of publication or study type. There was evidence of heterogeneity by sex (p<0.01), with a significant increase only for females (1.59, 1.16-2.19, n = 11). There was no heterogeneity by aspects of diagnosis, although the estimates were highest for definitions based on mortality or hospitalisation (1.40, 1.12-1.74, n = 11). However, there was significant heterogeneity (p<0.001) by numbers of cases, with larger ORs from studies of less than 50 cases (1.26, 0.83-1.92, n=11) and from studies of 50-149 cases (1.62, 1.35-1.96, n=9) than for studies of 150 or more cases (1.08, 0.97-1.20, n=13). There was no significant evidence of heterogeneity by extent of confounder adjustment, or by how never smokers or the exposure index were defined. For all these subgroup analyses, there was little evidence of publication bias, but evidence of heterogeneity in some subgroups.\n\nThe combined OR for severer COPD was significant (1.29, 1.10-1.52, n=7).\n\nThere was also evidence of a dose-response relationship, as shown in Table 5, with six of 11 studies investigating this reporting a statistically significant positive trend. Study 16 reported no trend in relation to the number of smokers in the household, but did report positive dose-response relationships for years of ETS exposure at home and at work. Study 19, which found no relationship with the main COPD outcome, also presented dose-response relationships for severer COPD, again finding no significant increase in risk with increasing exposure.\n\nFive studies also presented additional results for other indices of ETS exposure, as shown in Table 6. Four studies (16, 22, 23, 26) looked at exposure at work, all but study 23 also presenting results for combined exposure at home and at work. Study 5 produced a combined index of adulthood exposure at home or work, or during travel or leisure. Three studies (16, 23, 26) considered childhood ETS exposure, study 23 studying exposure from both the mother and the father, and also looking at parental smoking during pregnancy.\n\na Study types are CC = case-control, CS = cross-sectional, L = longitudinal. For longitudinal studies, number of years follow-up is shown\n\nb RRs from longitudinal studies are taken as being equivalent to ORs\n\nc NS p≥0.05, + p<0.05, ++ p<0.01\n\nd OR and/or CI estimated from data provided\n\ne Approximate estimates\n\nf Sum of smoking levels for all cohabitants\n\ng For husband smoking, there were 8 levels: never, former, current pipe/cigar, and current cigs/day 1-9, 10-19, 20, 21-39 and 40+. For wife smoking there were 7 levels, as for husband except with no level for pipe/cigar\n\nh Number of cases is for the exposed groups combined\n\ni Trend estimated from data provided\n\nj Sum of scores for exposure at home (0 = no exposure, 1 = <4 pack years, 2 = 4 to <8 pack years, 3 = ≥8 pack years) and at work (0 = no exposure, 1 = <5, 2 = 5 to <15, 3 = ≥15, calculated from (pack years x smokers x hours/day)/100\n\naStudy types are CC = case control, CS = cross-sectional, L = longitudinal. For longitudinal studies, number of years follow-up is shown\n\nbComparison is with no exposure of the type specified, except where indicated otherwise\n\ncRRs from longitudinal studies are taken as being equivalent to ORs\n\ndA = Any adult, C = Childhood and W = Workplace indicate estimates included in Table 7 meta-analysis\n\neComparison is with those with no exposure of any of the four types, or at most little exposure from one of them\n\nfOR and/or CI estimated from data provided\n\ngComparison is with those with <2 years of 40 hours per week exposure\n\nhCompares exposed at work only to unexposed. Excludes those exposed at home\n\niComparison group is subjects not exposed to ETS from any source\n\njFrom 65\n\nThe ORs for these other exposure indices are supported by Figure 3 (workplace) and Figure 4 (overall adult), while Table 7 presents the results of meta-analyses. Note that Figure 4, and the meta-analyses for overall adult exposure, consider not only the ORs indicated in Table 6, but also include estimates from Table 3 for those ten studies (4, 11, 12, 15, 17, 18, 19, 21, 25, 27) for which the exposure was at least from home and work.\n\nIndividual study estimates of the OR and its 95%CI are shown sorted in increasing order of OR. These are shown as numbers, and also graphically on a logarithmic scale. Random-effects estimates of ORs and 95%CIs are also shown. Studies are identified by the study number shown in Table 1. In the graphical representation, ORs are indicated by a square, with the area of the square proportional to the weight.\n\nIndividual study estimates of the OR and its 95%CI are shown sorted in increasing order of OR. These are shown as numbers, and also graphically on a logarithmic scale. Random-effects estimates of ORs and 95%CIs are also shown. Studies are identified by the study number shown in Table 1. In the graphical representation, ORs are indicated by a square, with the area of the square proportional to the weight.\n\na Definition of COPD as shown in the body of Table 1. Data as shown in Table 6 excluding severer COPD\n\nb Number of estimates in meta-analysis\n\nc Egger test p expressed as <0.001, <0.01, <0.05, <0.1 or NS (p≥0.1)\n\nd Degrees of freedom\n\ne p expressed as <0.001, <0.01, <0.05, <0.1 or NS (p≥0.1)\n\nf Index includes “home or workplace” or combined index of any adulthood exposure. Note that this meta-analysis not only includes those estimates marked with an A in Table 6, but also includes estimates from Table 3 for studies 4, 11, 12, 15, 17, 18, 19, 21, 25 and 27\n\ng Preferring exposure from the mother in study 23. Estimates would be 0.89 (0.74-1.08) fixed and 0.91 (0.70-1.18) random, preferring exposure from the father\n\nOf the four ORs included in the meta-analysis of COPD for exposure at work, two were above 1.00, one of borderline statistical significance, and two were below 1.00, the combined estimate being 1.12 (0.77-1.64). Note that in study 26 there was a choice of workplace OR estimates, with the meta-analysis including that for current exposure. Using estimates for previous or ever exposure would not have affected the conclusion that there was no clear relationship of COPD to workplace ETS exposure.\n\nOf the 17 ORs included in the meta-analysis for overall adult exposure, 12 were above 1.00, five significantly so, with one equal to 1.00, and four less than 1.00. The combined estimate of 1.20 (1.03-1.39) was also significantly increased.\n\nThere was no clear association of COPD with childhood ETS exposure, with none of the ORs shown in Table 6 being significant. Only two estimates could be included in the meta-analysis, giving an overall estimate of 0.88 (0.72-1.07).\n\nThere was no significant evidence of publication bias for workplace or adult exposure, the data being too limited to assess this for childhood exposure. However, there was evidence of heterogeneity (p<0.01) for overall adult ETS exposure.\n\nThe limited further dose-response data shown in Table 8 added little to the data already shown in Table 5.\n\na Study types are CC = case-control, CS = cross-sectional\n\nb NS = p≥0.05, + = p<0.05, ++ = p<0.01\n\nc Based on sum of 0 = not at all, 1 = little, 2 = average, 3 = a lot for each source of exposure\n\nd Trend estimated from data provided\n\n\nDiscussion\n\nWe rejected papers for various appropriate reasons. These included the following: failing to give results for an endpoint equivalent to COPD; giving results only for COPD exacerbation or prognosis; not presenting results for never smokers; describing studies without a control group; not presenting results for those unexposed to ETS; and presenting less useful results than reported in another publication.\n\nTwenty-eight epidemiological studies did qualify for inclusion, and from 33 estimates of the risk of COPD associated with ever having a spouse who smoked, or the nearest equivalent ETS exposure index available, random-effects meta-analysis gave a significantly increased OR estimate of 1.20 (1.08-1.34). There was also some evidence of dose-response. While the clear relationship of smoking with COPD31 makes it plausible that some effect will also be evident for ETS, one must emphasize that exposure is much less than from active smoking, as noted in the Introduction. Also, various limitations of the evidence, discussed below, make it difficult to be confident that a causal relationship exists.\n\nThough four studies involved more than 500 cases, with the maximum 1097 in study 15, as many as ten of the 28 studies involved less than 100 cases, the quite small number of cases making it difficult to detect potential effects reliably.\n\nThe observation that ORs are only modestly raised for studies with larger numbers of cases but are greater for smaller studies suggests the possibility of publication bias, with authors being more likely to report stronger relationships. However formal tests for publication bias54 showed no clear evidence of its existence. One must note, though, that various large longitudinal studies, e.g. 66–69, reported results relating ETS to smoking-related diseases such as lung cancer or heart disease, but did not do so for COPD. If any relationship had been seen, these studies might well have been reported.\n\nNo study validated the lifelong non-smoking status of their participants, although study 18 did verify current active and passive exposure in a random sample of participants by measuring urinary cotinine levels. As some current and past smokers deny smoking when interviewed70, and as the smoking habits of spouses or household members are clearly correlated47, misclassification of even a few ever smokers as never smokers can cause relevant bias71, especially when, as is the case with COPD, the association with smoking is strong31.\n\nAll the longitudinal studies considered involved follow-up for at least 12 years. Of the five studies, three (studies 3, 7 and 10) assumed spousal smoking was unchanged during follow-up, only studies 4 and 22 collecting information on smoking status at multiple time points.\n\nAll these studies only considered COPD deaths which occurred in the original study area.\n\nAlthough three case-control studies used population controls, the remaining three used control groups unlikely to be representative of the population from which the cases derived. Studies 6 and 14 used visitors to the hospital attended by the cases, and study 13 used as a control a person identified by the informant of a death as a “living person about the same age who was well known to the informant”, the informant then being asked about the lifestyle 10 years earlier of both decedent and control.\n\nOver half of the studies were of cross-sectional design, a design limited by difficulties in determining whether ETS exposure or disease onset occurred first.\n\nAs noted above, some studies made little or no adjustment for variables likely to differ between smoking and non-smoking households. Though ORs for the main exposure index did not vary significantly by extent of adjustment, it should be noted that adjustment for dietary variables and education explains a substantial part of the association of lung cancer with spousal smoking45. The same may be the case for COPD.\n\nDefinitions of COPD used were all consistent with the inclusion criteria. However, they still varied somewhat between study, further adding uncertainty to the meta-analysis results. Even given the inclusion criteria, there are doubts about the appropriateness of the diagnostic criteria used in some studies. In study 8, for example, the definition included asthma as well as chronic bronchitis and emphysema, the diagnosis being reported by the head of the household, and not necessarily made by a doctor.\n\nWhile random errors in determining ETS exposure led to underestimation of the relationship of COPD with ETS, errors may not be random. Twenty-three of the 28 studies considered were of case-control or cross-sectional design, where recall bias may exist if those with COPD tend to overestimate their ETS exposure compared to those without COPD. Exposure was generally not validated by biochemical markers or air measurements taken at home.\n\nOnly 15 studies (4, 5, 11, 12, 15–19, 21–23, 25–27) provided data on ETS exposure from sources other than the home. Five (4, 12, 17, 18, 27) presented results only for a combined household and workplace exposure index, with a further five (11, 15, 19, 21, 25) only presenting results for total exposure irrespective of location, results used in our analyses as the nearest equivalent which was available to smoking by the spouse or household member. While there are far less available data on risk of COPD from ETS exposure specifically in the workplace or in childhood than on smoking in the home, the available data show no clear relationship of risk with these less studied exposure indices.\n\nA review in 200772 considered that “ETS exposure may be an important cause of COPD”. However, this conclusion was based on only six studies, one examining absolute risk of COPD in relation to changes in tobacco consumption, and one comparing lung function of employees in bars and restaurants before and after a smoking ban. Also it seemed that at least some of the others considered were not restricted to never smokers.\n\nA review in 201060 meta-analysed results from 12 studies and gave an overall estimate of 1.56 (1.40-1.74), somewhat higher than our estimate. Not all of the studies included were of COPD, some being based on chronic bronchitis symptoms. Also, some studies were based on current non-smokers rather than on lifelong never smokers.\n\nIn 2013, Bentayeb et al.61 reviewed evidence on indoor air pollution and respiratory health in those aged over 65 years. After considering 33 papers (only one16 presenting relevant results on ETS and COPD risk in non-smokers), they reported that the most consistent relationship found was between ETS exposure and COPD risk. However, the findings did not allow causal inference due to heterogeneity of the studies considered, measurement errors in exposure assessment, variable outcome definition, and lack of information on lifetime exposure to air pollution. The authors concluded that more investigations are needed to understand the relationship of indoor air pollution to respiratory health in the elderly.\n\nA review in 201473 reached similar conclusions, the authors stating that “second-hand exposure to tobacco smoke has also been shown to be associated with the risk of COPD, although more robust evidence needs to be generated”. These conclusions were derived from only eight studies, some concerned with respiratory symptoms rather than COPD. Also, one study did not restrict any analyses to never smokers.\n\nA review in 201562 included only five studies in the meta-analyses. The estimated risk of COPD in ETS-exposed participants was higher than we estimated, being 1.66 (1.38-2.00) for both sexes combined, 1.50 (0.96-2.28) for males and 2.17 (1.48-3.18) for females. However, these estimates were based on, respectively, three, one and one estimates, the authors examining three further studies but not including them in their meta-analysis due to low study quality. However, two of the studies they did include were not based on lifelong never smokers. Many other studies that might have been included were not. The authors noted that “the few existing studies on second-hand smoke exposure and COPD differ considerably, although the results indicate a positive association” and that “further research is needed, to provide more adequate primary studies which account for confounding and other biases”.\n\nA review in 201663 of “the effects of smoking on respiratory health” also considered effects of ETS exposure. However, only three studies were cited, two not satisfying our inclusion criteria. Noting the variability in the results, the authors only pointed to the need for additional studies.\n\nIt is clear that the present review includes far more studies relating ETS exposure to risk of COPD in never smokers than considered by others.\n\n\nConclusion\n\nThe evidence may be regarded as suggestive of a possible effect of ETS exposure on risk of COPD, especially given the strong association of smoking with the disease. However, given the marginal significance of the meta-analyses, the absence of well-designed and fully reported large studies, and the limitations noted above, the evidence must be regarded as inadequate to infer a causal relationship. More definitive studies are required to reach a firmer conclusion.", "appendix": "Competing interests\n\n\n\nPNL, Director of P.N. Lee Statistics and Computing Ltd., is an independent consultant in statistics and an advisor in epidemiology to various tobacco companies, including the sponsors of this study. KJC is an employee of, and AJT a consultant to, P.N. Lee Statistics and Computing Ltd. BAF and JSH were also employees when the work was conducted.\n\n\nGrant information\n\nJapan Tobacco International S.A. supported this work by a grant to P. N. Lee Statistics and Computing Ltd.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Japan Tobacco International S.A. for supporting the work described. The opinions and conclusions of the authors are their own, and do not necessarily reflect the position of Japan Tobacco International S.A. We also thank Diana Morris and Yvonne Cooper for assistance in typing various drafts of the paper and obtaining relevant literature, and all the researchers who published the reports which formed the basis of our work.\n\n\nSupplementary material\n\nSupplementary file 1: Rejected studies: In preparing the tables and figures in this document, certain papers that might be thought to provide relevant data have not been referred to. For each of these papers, this appendix notes the authors, date of publication and country and the reasons for not referring to them.\n\nClick here to access the data.\n\nSupplementary file 2 : PRISMA checklist\n\nClick here to access the data.\n\nSupplementary file 3: Forest plots in Excel\n\nClick here to access the data.\n\n\nReferences\n\nLebowitz MD, Burrows B: Respiratory symptoms related to smoking habits of family adults. Chest. 1976; 69(1): 48–50. PubMed Abstract \| Publisher Full Text\n\nComstock GW, Meyer MB, Helsing KJ, et al.: Respiratory effects on household exposures to tobacco smoke and gas cooking. Am Rev Respir Dis. 1981; 124(2): 143–148. PubMed Abstract\n\nHirayama T: Lung cancer in Japan: effects of nutrition and passive smoking. In: Mizell M and Correa P, editors. Lung cancer: causes and prevention. Proceedings of the International Lung Cancer Update Conference; March 3–5, 1983; New Orleans, Louisiana. Deerfield Beach, Florida: Verlag Chemie International, Inc, 1984; 175–195. Data clarifications appear in Passive smoking [Letter]. N Z Med J. 1990; 103(883): 54. PubMed Abstract\n\nKrzyzanowski M, Jedrychowski W, Wysocki M: Factors associated with the change in ventilatory function and the development of chronic obstructive pulmonary disease in a 13-year follow-up of the Cracow Study. Risk of chronic obstructive pulmonary disease. Am Rev Respir Dis. 1986; 134(5): 1011–1019. PubMed Abstract \| Publisher Full Text\n\nLee PN, Chamberlain J, Alderson MR: Relationship of passive smoking to risk of lung cancer and other smoking-associated diseases. Br J Cancer. 1986; 54(1): 97–105. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKalandidi A, Trichopoulos D, Hatzakis A, et al.: Passive smoking and chronic obstructive lung disease [Letter]. Lancet. 1987; 2(8571): 1325–1326. PubMed Abstract \| Publisher Full Text\n\nSandler DP, Comstock GW, Helsing KJ, et al.: Deaths from all causes in non-smokers who lived with smokers. Am J Public Health. 1989; 79(2): 163–167. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDayal HH, Khuder S, Sharrar R, et al.: Passive smoking in obstructive respiratory disease in an industrialized urban population. Environ Res. 1994; 65(2): 161–171. PubMed Abstract \| Publisher Full Text\n\nForastiere F, Mallone S, Lo Presti E, et al.: Characteristics of nonsmoking women exposed to spouses who smoke: epidemiologic study on environment and health in women from four Italian areas. Environ Health Perspect. 2000; 108(12): 1171–7. PubMed Abstract \| Free Full Text\n\nEnstrom JE, Kabat GC: Environmental tobacco smoke and tobacco related mortality in a prospective study of Californians, 1960–98. BMJ. 2003; 326(7398): 1057. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Marco R, Accordini S, Cerveri I, et al.: An international survey of chronic obstructive pulmonary disease in young adults according to GOLD stages. Thorax. 2004; 59(2): 120–125. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCelli BR, Halbert RJ, Nordyke RJ, et al.: Airway obstruction in never smokers: results from the Third National Health and Nutrition Examination Survey. Am J Med. 2005; 118(12): 1364–1372. PubMed Abstract \| Publisher Full Text\n\nMcGhee SM, Ho SY, Schooling M, et al.: Mortality associated with passive smoking in Hong Kong. BMJ. 2005; 330(7486): 287–288. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSezer H, Akkurt I, Guler N, et al.: A case-control study on the effect of exposure to different substances on the development of COPD. Ann Epidemiol. 2006; 16(1): 59–62. PubMed Abstract \| Publisher Full Text\n\nXu F, Yin X, Shen H, et al.: Better understanding the influence of cigarette smoking and indoor air pollution on chronic obstructive pulmonary disease: a case-control study in Mainland China. Respirology. 2007; 12(6): 891–897. PubMed Abstract \| Publisher Full Text\n\nYin P, Jiang CQ, Cheng KK, et al.: Passive smoking exposure and risk of COPD among adults in China: the Guangzhou Biobank Cohort Study. Lancet. 2007; 370(9589): 751–757. PubMed Abstract \| Publisher Full Text\n\nZhou Y, Wang C, Yao W, et al.: COPD in Chinese nonsmokers. Eur Respir J. 2009; 33(3): 509–518. PubMed Abstract \| Publisher Full Text\n\nWu CF, Feng NH, Chong IW, et al.: Second-hand smoke and chronic bronchitis in Taiwanese women: a health-care based study. BMC Public Health. 2010; 10: 44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJordan RE, Cheng KK, Miller MR, et al.: Passive smoking and chronic obstructive pulmonary disease: cross-sectional analysis of data from the Health Survey for England. BMJ Open. 2011; 1(2): e000153. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLamprecht B, McBurnie MA, Vollmer WM, et al.: COPD in never smokers: results from the population-based burden of obstructive lung disease study. Chest. 2011; 139(4): 752–763. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen R: Association of environmental tobacco smoke with dementia and Alzheimer's disease among never smokers. Alzheimers Dement. 2012; 8(6): 590–595. PubMed Abstract \| Publisher Full Text\n\nHe Y, Jiang B, Li LS, et al.: Secondhand smoke exposure predicted COPD and other tobacco-related mortality in a 17-year cohort study in China. Chest. 2012; 142(4): 909–918. PubMed Abstract \| Publisher Full Text\n\nWaked M, Salame J, Khayat G, et al.: Correlates of COPD and chronic bronchitis in nonsmokers: data from a cross-sectional study. Int J Chron Obstruct Pulmon Dis. 2012; 7: 577–585. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoreira MA, Barbosa MA, Jardim JR, et al.: Chronic obstructive pulmonary disease in women exposed to wood stove smoke. Rev Assoc Med Bras (1992). 2013; 59(6): 607–13. PubMed Abstract \| Publisher Full Text\n\nEze IC, Schaffner E, Zemp E, et al.: Environmental tobacco smoke exposure and diabetes in adult never-smokers. Environ Health. 2014; 13: 74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHagstad S, Bjerg A, Ekerljung L, et al.: Passive smoking exposure is associated with increased risk of COPD in never smokers. Chest. 2014; 145(6): 1298–1304. PubMed Abstract \| Publisher Full Text\n\nKim WJ, Song JS, Park DW, et al.: The effects of secondhand smoke on chronic obstructive pulmonary disease in nonsmoking Korean adults. Korean J Intern Med. 2014; 29(5): 613–619. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTan WC, Sin DD, Bourbeau J, et al.: Characteristics of COPD in never-smokers and ever-smokers in the general population: results from the CanCOLD study. Thorax. 2015; 70(9): 822–829. PubMed Abstract \| Publisher Full Text\n\nAdvisory Committee to the Surgeon General of the Public Health Service: Smoking and health. Report of the Advisory Committee to the Surgeon General of the Public Health Service. Public Health Service Publication No. 1103. Vol Washington DC: US Department of Health, Education, and Welfare; Public Health Service, 1964; 387. Reference Source\n\nUS Surgeon General: The Health Consequences of Smoking: A Report of the Surgeon General. Vol Atlanta, Georgia: US Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health, 2004; 911. PubMed Abstract\n\nForey BA, Thornton AJ, Lee PN: Systematic review with meta-analysis of the epidemiological evidence relating smoking to COPD, chronic bronchitis and emphysema. BMC Pulm Med. 2011; 11: 36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInternational Agency for Research on Cancer: Tobacco smoke and involuntary smoking. IARC Monogr Eval Carcinog Risks Hum Lyon, France: IARC. 2004; 83: 1452. Reference Source\n\nOffice of Population Censuses and Surveys: Health survey for England 1994. Volume I: Findings. Volume II: Survey methodology & documentation. Series HS no. 4. Colhoun H and Prescott-Clarke P, editors. Vol London: HMSO, 1996; 607. Reference Source\n\nZiegler RG, Mason TJ, Stemhagen A, et al.: Dietary carotene and vitamin A and risk of lung cancer among white men in New Jersey. J Natl Cancer Inst. 1984; 73(6): 1429–1435. PubMed Abstract \| Publisher Full Text\n\nPirkle JL, Flegal KM, Bernert JT, et al.: Exposure of the US population to environmental tobacco smoke: the Third National Health and Nutrition Examination Survey, 1988 to 1991. JAMA. 1996; 275(16): 1233–1240. PubMed Abstract\n\nBenowitz NL, Bernert JT, Caraballo RS, et al.: Optimal serum cotinine levels for distinguishing cigarette smokers and nonsmokers within different racial/ethnic groups in the United States between 1999 and 2004. Am J Epidemiol. 2009; 169(2): 236–248. PubMed Abstract \| Publisher Full Text\n\nPhillips K, Bentley MC, Howard DA, et al.: Assessment of air quality in Stockholm by personal monitoring of nonsmokers for respirable suspended particles and environmental tobacco smoke. Scand J Work Environ Health. 1996; 22(Suppl 1): 1–24. PubMed Abstract\n\nPhillips K, Bentley MC, Howard DA, et al.: Assessment of air quality in Barcelona by personal monitoring of nonsmokers for respirable suspended particles and environmental tobacco smoke. Environ Int. 1997; 23(2): 173–196. Publisher Full Text\n\nPhillips K, Howard DA, Bentley MC, et al.: Assessment of air quality in Kuala Lumpur by personal monitoring of nonsmokers at home and in the workplace by reference to respirable suspended particles (RSP) and environmental tobacco smoke (ETS). In: Gee IL and Leslie GB, editors. Indoor and built environment problems in Asia. Proceedings of the Conference; 4th & 5th September 1997; Kuala Lumpur, Malaysia. Rothenfluh, Switzerland: The International Society of the Built Environment. 1997; 151–159.\n\nPhillips K, Howard DA, Bentley MC, et al.: Assessment by personal monitoring of respirable suspended particles and environmental tobacco smoke exposure for non-smokers in Sydney, Australia. Indoor Built Environ. 1998; 7(4): 188–203. Publisher Full Text\n\nPhillips K, Howard DA, Bentley MC: Assessment of environmental tobacco smoke and respirable suspended particle exposures for nonsmokers in Lisbon by personal monitoring. Environ Int. 1998; 24(3): 301–324. Publisher Full Text\n\nPhillips K, Howard DA, Bentley MC, et al.: Measured exposures by personal monitoring for respirable suspended particles and environmental tobacco smoke of housewives and office workers resident in Bremen, Germany. Int Arch Occup Environ Health. 1998; 71(3): 201–212. PubMed Abstract \| Publisher Full Text\n\nPhillips K, Bentley MC, Howard DA, et al.: Assessment of environmental tobacco smoke and respirable suspended particle exposures for nonsmokers in Prague using personal monitoring. Int Arch Occup Environ Health. 1998; 71(6): 379–390. PubMed Abstract \| Publisher Full Text\n\nPhillips K, Howard DA, Bentley MC, et al.: Environmental tobacco smoke and respirable suspended particle exposures for non-smokers in Beijing. Indoor Built Environ. 1998; 7(5–6): 254–269. Publisher Full Text\n\nLee PN, Fry JS, Forey B, et al.: Environmental tobacco smoke exposure and lung cancer: a systematic review. World J Metaanal. 2016; 4(2): 10–43. Publisher Full Text\n\nUS Surgeon General: The health consequences of involuntary exposure to tobacco smoke. A report of the Surgeon General. Vol Atlanta, Georgia: US Department of Health and Human Services, Centers for Disease Control and Prevention, Coordinating Center for Health Promotion, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health, 2006; 727. Reference Source\n\nLee PN: Environmental tobacco smoke and mortality. A detailed review of epidemiological evidence relating environmental tobacco smoke to the risk of cancer, heart disease and other causes of death in adults who have never smoked. Vol Basel: Karger. 1992; 224. Publisher Full Text\n\nGlobal Initiative for Chronic Obstructive Disease: Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. 2006. Executive summary. Vol Medical Communications Resources, Inc, 2006. Reference Source\n\nLiberati A, Altman DG, Tetzlaff J, et al.: The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate healthcare interventions: explanation and elaboration. BMJ. 2009; 339: b2700. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMorris JA, Gardner MJ: Calculating confidence intervals for relative risks (odds ratios) and standardised ratios and rates. Br Med J (Clin Res Ed). 1988; 296(6632): 1313–1316. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHamling J, Lee P, Weitkunat R, et al.: Facilitating meta-analyses by deriving relative effect and precision estimates for alternative comparisons from a set of estimates presented by exposure level or disease category. Stat Med. 2008; 27(7): 954–970. PubMed Abstract \| Publisher Full Text\n\nFleiss JL, Gross AJ: Meta-analysis in epidemiology, with special reference to studies of the association between exposure to environmental tobacco smoke and lung cancer: a critique. J Clin Epidemiol. 1991; 44(2): 127–139. PubMed Abstract \| Publisher Full Text\n\nHiggins JP, Thompson SG, Deeks JJ, et al.: Measuring inconsistency in meta-analyses. BMJ. 2003; 327(7414): 557–560. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEgger M, Davey Smith G, Schneider M, et al.: Bias in meta-analysis detected by a simple, graphical test. BMJ. 1997; 315(7109): 629–634. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee PN: Uses and abuses of cotinine as a marker of tobacco smoke exposure. In: Gorrod JW and Jacob P, III: Analytical determination of nicotine and related compounds and their metabolites. Amsterdam: Elsevier, 1999; 669–719. Publisher Full Text\n\nTrédaniel J, Boffetta P, Saracci R, et al.: Exposure to environmental tobacco smoke and risk of lung cancer: the epidemiological evidence. Eur Respir J. 1994; 7(10): 1877–88. PubMed Abstract \| Publisher Full Text\n\nCoultas DB: Health effects of passive smoking. 8. Passive smoking and risk of adult asthma and COPD: an update. Thorax. 1998; 53(5): 381–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJaakkola MS, Jaakkola JJ: Effects of environmental tobacco smoke on the respiratory health of adults. Scand J Work Environ Health. 2002; 28(Suppl 2): 52–70. PubMed Abstract\n\nEisner MD, Balmes J, Katz PP, et al.: Lifetime environmental tobacco smoke exposure and the risk of chronic obstructive pulmonary disease. Environ Health. 2005; 4(1): 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEisner MD, Anthonisen N, Coultas D, et al.: An official American Thoracic Society public policy statement: Novel risk factors and the global burden of chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2010; 182(5): 693–718. PubMed Abstract \| Publisher Full Text\n\nBentayeb M, Simoni M, Norback D, et al.: Indoor air pollution and respiratory health in the elderly. J Environ Sci Health A Tox Hazard Subst Environ Eng. 2013; 48(14): 1783–9. PubMed Abstract \| Publisher Full Text\n\nFischer F, Kraemer A: Meta-analysis of the association between second-hand smoke exposure and ischaemic heart diseases, COPD and stroke. BMC Public Health. 2015; 15: 1202. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJayes L, Haslam PL, Gratziou CG, et al.: SmokeHaz: Systematic Reviews and Meta-analyses of the Effects of Smoking on Respiratory Health. Chest. 2016; 150(1): 164–79. PubMed Abstract \| Publisher Full Text\n\nFry JS, Lee PN: Revisiting the association between environmental tobacco smoke exposure and lung cancer risk. II. Adjustment for the potential confounding effects of fruit, vegetables, dietary fat and education. Indoor Built Environ. 2001; 10(1): 20–39. Publisher Full Text\n\nHagstad S, Ekerljung L, Lindberg A, et al.: COPD among non-smokers - report from the obstructive lung disease in Northern Sweden (OLIN) studies. Respir Med. 2012; 106(7): 980–988. PubMed Abstract \| Publisher Full Text\n\nLeVois ME, Layard MW: Publication bias in the environmental tobacco smoke/coronary heart disease epidemiologic literature. Regul Toxicol Pharmacol. 1995; 21(1): 184–191. PubMed Abstract \| Publisher Full Text\n\nSteenland K, Thun M, Lally C, et al.: Environmental tobacco smoke and coronary heart disease in the American Cancer Society CPS-II cohort. Circulation. 1996; 94(4): 622–628. PubMed Abstract \| Publisher Full Text\n\nJee SH, Ohrr H, Kim IS: Effects of husbands' smoking on the incidence of lung cancer in Korean women. Int J Epidemiol. 1999; 28(5): 824–828. PubMed Abstract \| Publisher Full Text\n\nWang A, Kubo J, Luo J, et al.: Active and passive smoking in relation to lung cancer incidence in the Women's Health Initiative Observational Study prospective cohort. Ann Oncol. 2015; 26(1): 221–230. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee PN, Forey BA: Misclassification of smoking habits as determined by cotinine or by repeated self-report - a summary of evidence from 42 studies. J Smoking-Related Dis. 1995; 6: 109–129. Reference Source\n\nLee PN, Forey BA: Misclassification of smoking habits as a source of bias in the study of environmental tobacco smoke and lung cancer. Stat Med. 1996; 15(6): 581–605. PubMed Abstract \| Publisher Full Text\n\nReardon JZ: Environmental tobacco smoke: respiratory and other health effects. Clin Chest Med. 2007; 28(3): 559–73, vi. PubMed Abstract \| Publisher Full Text\n\nSalvi S: Tobacco smoking and environmental risk factors for chronic obstructive pulmonary disease. Clin Chest Med. 2014; 35(1): 17–27. PubMed Abstract \| Publisher Full Text" }	[ { "id": "33219", "date": "11 May 2018", "name": "Sara Maio", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe review could be of interest but some some major revisions are needed. In particular, it would be important to update the papers selected for the revision because many other scientific evidences were published after June 2016. Moreover, too many aspects and subsets are taken into account; they did not add information and they make the paper hard to read and the message not clear.\n\nAs regards to the subsets chosen for the meta-analysis, it would be important to take into account the publication period before and after the smoking ban, because it determined a variation in the exposure of the people to passive smoke and the related effects.\n\nIn the discussion, section \"publication bias\", it was reported that \" One must note, though, that various large longitudinal studies, e.g. 66–69, reported results relating ETS to smokingrelated diseases such as lung cancer or heart disease, but did not do so for COPD. If any relationship had been seen, these studies might well have been reported.\". The reviewer thinks that this sentence is too strict and not supported by evidences.\n\nOverall the discussion seems to set up versus a negative approach against the results of the other published reviews; on the other side, it did not discuss the results regarding the negative effect of exposure to ETS found using meta-analyses. The reviewer thinks that it is not to overlook the results about the dose-response evidence or the findings of the whole sample meta-analysis (OR 1.20) etc...\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [ { "c_id": "3649", "date": "15 May 2018", "name": "Peter Lee", "role": "Author Response", "response": "Dear Dr Maio, I thank you for your comments which I respond to below on behalf of my co-authors. The text of the paper has not yet been altered as the editorial team advise that I wait for the additional referee reports before doing so. Updating selected papers You say that \"many other scientific evidences were published after June 2016. However, we have updated our searches to cover more recent papers, and only found one paper which satisfied the inclusion criteria. This was a report by Ukawa et al in 2017 (International Journal of Public Health, vol 62, pp 489-494) which presented results for at home passive smoking exposure from the Japan Collaborative cohort study, based only on 33 cases. Rather than updating the whole range of analysis results, we intend simply to refer to this additional study, and the effect it had on the overall effect estimate, in a comment at the end of the discussion section. If you think there are other important papers we have missed please let us know what they are. Too many aspects and subsets are taken into account We have published a number of previous reviews of the relationship of passive smoking to other diseases, and this style has never before been criticized. In our view it is important to fully describe how the association of interest varies by the source of exposure and by study characteristics, and also by the definition of disease. One cannot get a good insight without these details. Taking into account the publication period before and after the smoking ban You say \"the smoking ban\" but there are many smoking bans, different in type and different in timing. In the US for example different states, and different locations within states, brought in bans at different times. When one also considers the long latent period of COPD, with deaths post-ban perhaps due to exposures pre-ban, and the fact that in some studies some COPD cases occur pre-ban and some post-ban, we did not consider it useful to attempt the required analysis. Publication bias We made the point (also made in other passive smoking reviews) that some large cohort studies are known to have published positive relationships relating passive smoking to other diseases when they did not publish results relating passive smoking to COPD. Surely it is quite likely that they did not find a.positive relationship for COPD? In my view large cohort studies ought to publish passive smoking results for all diseases with sufficient cases, but often they do not. Our comment is supported by the evidence as to what has and has not been published - though this does not prove that all such studies found no positive association with COPD, the likelihood is there. The argument is similar to the general one for publication bias. We don't generally have evidence that papers showing no association are less likely to be submitted or accepted than papers finding an association, but it is highly plausible. Negative approach against other reviews We state the reasons why these other reviews are limited. Overlooking evidence suggesting an association, such as the dose-response results and the overall meta-analysis results In paragraph 2 of the discussion we refer to the overall meta-analysis results and the dose-response results, and then go on to discuss why these results are only suggestive of a causal relationship. The overall association of 1.20 with passive smoking at home, though highly statistically significant, is quite small in magnitude, and it is certainly possible that it may be explicable in terms of bias. In our review of passive smoking and lung cancer (World Journal of Meta-Analysis, 2017, 4, 10-43) we were able to demonstrate quite clearly that a similar sized association could plausibly be explained by a combination of uncontrolled confounding and misclassification of smoking status. Though the data for COPD are not extensive enough to readily allow such adjustments, we would be extremely nervous in saying that the association is more than suggestive of a causal relationship. Nevertheless, we will look again at the wording we have used and try to make our argument clearer. Conclusions drawn not adequately supported by the results presented This really relates to the previous point. We believe our conclusions are supported by the results. We would be happy to hear your reactions to our replies. Yours sincerely Peter Lee (and co-authors)" } ] }, { "id": "55818", "date": "22 Nov 2019", "name": "Yousser Mohammad", "expertise": [ "Reviewer Expertise Passive smoking", "Chronic Respiratory Diseases", "impact of conflict" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe review is interesting, because it highlights methodological issues and gaps in surveys assessing the impact of environmental tobacco smoke on COPD incidence. These gaps and inconsistencies in surveys, lead to inadequacy on assessing the impact, and showing non-confirmative results (OR, RR, etc) on the association between ETS exposure and COPD.\nI recommend it for indexing with minor changes.\nHowever, the article should address three additional paragraphs or remark on:\nPublic health message of awareness, it should address that, even if non association is found between ETS and COPD, it is still that ETS kills 90,0000 people per year and it is a global public health issue. Haahtela et al. Helsinki by nature. The nature step for respiratory health,Clinical and Transitional Allergy1.\n\nShould mention water pipe because it is wide spreading: Yousser Mohammad, Rafea Shaaban, Bassam Abou Al-Zahab, Nikolai Khaltaev, Jean Bousquet, Basim Dubaybo. Impact of active and passive smoking as risk factors for asthma and COPD in women presenting to primary care in Syria: first report by the WHO-GARD survey group2. WHO Advisory note 2015: waterpipe tobacco smoking: health effects, research needs and recommended actions by regulators – 2nd ed. World Health Organization. II.WHO Study Group on Tobacco Product Regulation. ISBN 978 92 4 150846 9 (NLM classification: QV 137)3.\n\nShould discuss the difference in the quality of ETS\nNot only nicotine but how the smoke is modified in the air and becoming may be more harmful to small airways:\nLower ignition temperature Particles become smaller Higher proportion of CO, VOC\nRef found in the article: Mohammad Y. Passive smoking interference with wheezing and asthma Short Review of current knowledge4.\nWirth et al. Respiratory diseases related to passive smoking5.\n\nBeside that, in one study6 the duration of exposure is 2 weeks, maybe we should omit it.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "5100", "date": "19 Dec 2019", "name": "Peter Lee", "role": "Author Response", "response": "Reply to Yousser Mohammad Dr Mohammad suggests that I should cite a statement by WHO that ETS is responsible for about 900,000 deaths per year from all causes combined. We would rather not do this for two reasons. First, the paper is specifically about ETS and COPD, so does not need to stray into the relationship between ETS and other causes of death. Secondly, estimation of the effect of ETS on overall mortality is extremely complex and citation of a single estimate is questionable. We have in fact published widely on the evidence relating ETS to other diseases, such as lung cancer (Lee et al., 2016a; Lee et al., 2002), other cancers (Lee, 2002; Lee and Hamling, 2006; Lee and Hamling, 2016; Lee et al., 2016b), stroke (Lee and Forey, 2006; Lee et al., 2017b), heart disease (Lee et al., 2017a), and asthma (Lee and Forey, 2007), and find little evidence of an effect as large as WHO claims. For lung cancer, for example, we concluded (Lee et al., 2017b) that any causal relationship is not convincingly demonstrated, as most, if not all, of the relationship with ETS can be explained by confounding adjustment and misclassification correction.Dr Mohammad also suggests that I mention waterpipes. We would prefer not to do this as the paper is about ETS exposure from conventional cigarettes and as we have not formally reviewed the evidence relating to waterpipes. We have made it clearer in the introduction that the paper concerns ETS from cigarettes.He also suggests that we should discuss differences in the quality of ETS. We assume that he is pointing out that ETS from various products may not have the same composition or effects as from conventional cigarettes. But we are not concerned with ETS from products other than cigarettes.He suggests that we refer to evidence relating ETS to wheezing and asthma. While the paper he cites does not actually mention COPD, we do now include a statement in the text at the end of paragraph 2 of the discussion. ReferencesLee, P.N., 2002. Environmental tobacco smoke and cancer of sites other than the lung in adult non-smokers. Food Chem. Toxicol. 40, 747-766. Lee, P.N., Forey, B.A., 2006. Environmental tobacco smoke exposure and risk of stroke in nonsmokers: a review with meta-analysis. J. Stroke Cerebrovasc. Dis. 15, 5, 190-201. Lee, P.N., Forey, B.A., 2007. The role of environmental tobacco smoke in asthma induction and exacerbation in children and adults. Nova Science Publishers, Inc., New York. Lee, P.N., Forey, B.A., Hamling, J.S., Thornton, A.J., 2017a. Environmental tobacco smoke exposure and heart disease: A systematic review. World J. Metaanal. 5, 2, 14-40. DOI:10.13105/wjma.v5.i2.14. Lee, P.N., Fry, J.S., Forey, B., Hamling, J.S., Thornton, A.J., 2016a. Environmental tobacco smoke exposure and lung cancer: a systematic review. World J. Metaanal. 4, 2, 10-43. DOI:10.13105/wjma.v4.i2.10. Lee, P.N., Fry, J.S., Forey, B.A., 2002. Revisiting the association between environmental tobacco smoke exposure and lung cancer risk. V. Overall conclusions. Indoor Built Environ. 11, 59-82. DOI:10.1177/1420326X0201100202. Lee, P.N., Hamling, J., 2006. Environmental tobacco smoke exposure and risk of breast cancer in nonsmoking women: a review with meta-analyses. Inhal. Toxicol. 18, 14, 1053-1070. Lee, P.N., Hamling, J.S., 2016. Environmental tobacco smoke exposure and risk of breast cancer in nonsmoking women. An updated review and meta-analysis. Inhal. Toxicol. 28, 10, 431-54. DOI:10.1080/08958378.2016.1210701. Lee, P.N., Thornton, A.J., Forey, B.A., Hamling, J.S., 2017b. Environmental tobacco smoke exposure and risk of stroke in never smokers: An updated review with meta-analysis. J. Stroke Cerebrovasc. Dis. 26, 1, 204-216. DOI:10.1016/j.jstrokecerebrovasdis.2016.09.011. Lee, P.N., Thornton, A.J., Hamling, J.S., 2016b. Epidemiological evidence on environmental tobacco smoke and cancers other than lung or breast. Regul. Toxicol. Pharmacol. 80, 134-163. DOI:10.1016/j.yrtph.2016.06.012." } ] } ]	1	https://f1000research.com/articles/7-146
https://f1000research.com/articles/8-560/v1	26 Apr 19	{ "type": "Method Article", "title": "Construction and optimization of a ‘NG Morbidostat’ - An automated continuous-culture device for studying the pathways towards antibiotic resistance in Neisseria gonorrhoeae", "authors": [ "Els Verhoeven", "Said Abdellati", "Patrick Nys", "Jolein Gyonne Elise Laumen", "Irith De Baetselier", "Tania Crucitti", "Chris Kenyon", "Said Abdellati", "Patrick Nys", "Irith De Baetselier", "Tania Crucitti", "Chris Kenyon" ], "abstract": "To obtain a detailed picture of the dynamics of antibiotic resistance development in Neisseria gonorrhoeae, we built a morbidostat according to the protocol of Toprak et al., adjusted to the specific characteristics required for the growth of N. gonorrhoeae. In this article we describe the adaptations, specifications and the difficulties we encountered during the construction and optimization of the NG morbidostat. As a proof of concept, we conducted a morbidostat experiment by increasing concentrations of azithromycin in response to bacterial growth. We started the experiment with two N. gonorrhoeae reference strains WHO-F and WHO-X. These strains were grown in 12 mL GC Broth supplemented with IsoVitaleX™ (1%) and vancomycin, colistin, nystatin, trimethoprim (VCNT) selective supplement for 30 days in a 6% CO2 environment at 36°C. Samples of the cultures were taken 2-3 times a week and minimal inhibitory concentrations (MICs) of azithromycin were determined using E-test. The initial MICs of WHO-F and WHO-X were 0.125 µg/mL and 0.25 µg/mL, respectively. In less than 30 days, we were able to induce high level azithromycin resistance in N. gonorrhoeae, with a 750 and 1000 fold increase in MIC for WHO-F and WHO-X, respectively.", "keywords": [ "morbidostat", "Neisseria gonorrhoeae", "antibiotics", "macrolide resistance", "antimicrobial resistance", "sexual transmitted infection" ], "content": "Introduction\n\nGonorrhoea is a sexual transmitted infection (STI) caused by the obligate human pathogen Neisseria gonorrhoeae (gonococcus), a Gram-negative diplococcus1,2. Transmission of the gonococcus occurs primarily by direct contact between the mucosal membranes in the urogenital tract, anal canal and oropharynx, usually during sexual activity2.\n\nGonorrhoea is one of the most commonly reported STIs3. The World Health Organization (WHO) estimated an incidence of 87 million new cases for gonorrhoea (individuals aged 15–49 years) in 2016, worldwide4. An increase in cases of gonorrhoea has been reported in several European countries: 11% rise from 2014 to 2015 in the United Kingdom5 and a doubling of cases in 2013 and 2015 among men who have sex with men (MSM) in France6.\n\nThere is a growing global concern about antimicrobial resistance in N. gonorrhoeae, with resistance reported to almost all antimicrobials previously and currently available for treatment7. In response to this concern, the Centers for Disease Control and Prevention (CDC) and the European guidelines for treatment of gonorrhoea introduced dual antimicrobial therapy with azithromycin (oral) and ceftriaxone (injectable) for uncomplicated gonorrhoea in 20122,8. Dual therapy was also recommended by WHO in 20169. However, the prevalence of ceftriaxone and azithromycin-resistant strains is increasing in certain areas10,11.\n\nTherefore, more research is required to investigate how N. gonorrhoeae develops antibiotic resistance so rapidly. A better understanding of the pathways to resistance may enable novel strategies to prevent the emergence of resistance in the future12. In order to evaluate the dynamics of resistance development, Toprak et al.13 developed a microbial culture device called a ‘morbidostat’. A morbidostat is a bioreactor that continuously monitors bacterial growth and adjusts antibiotic concentration to induce bacterial resistance against the drug13,14. Experiments using a morbidostat can give us insights into the evolution of resistance and the nature, order and speed at which mutations arise. Whole-genome sequencing can be used to characterize the sequential mutation steps in the resistance genesis in great detail14,15.\n\nThe aim of this study was to build a morbidostat according to the protocol of Toprak et al13. We report here on the construction, optimization and a proof of concept of the NG morbidostat, a morbidostat adjusted to the specific characteristics required for the growth of N. gonorrhoeae.\n\n\nMethods\n\nThe morbidostat measures growth rates based on the intensity of back-scattered light through a bacterial suspension using an optical detection system. The bacteria are suspended in a fixed volume of liquid medium with continuous stirring. At fixed time intervals, the culture is diluted with a fixed volume. Depending on turbidity measurements and growth rate, an algorithm defines dilution with fresh medium or fresh medium containing antibiotic. Fresh medium with antibiotic is injected if the turbidity exceeds a threshold and when the net growth rate of the bacteria is positive. This threshold is set in the mid-log phase of the growth curve. At this point, there is a high metabolic activity present in the bacteria and the population is not nutrient limited. To allow N. gonorrhoeae to adapt to the environment, a threshold for fresh medium is set as well. Over time, the concentration of antibiotic added to these vials increases and drug resistance in bacteria evolves. During the entire experiment, the volume in the morbidostat culture vials is kept constant using a 16-channel suction pump (Ismatec, ISM938D). Morbidostat experiments are continued until a diminishing rate of increase in drug resistance is observed13,14.\n\nThe NG morbidostat consisted of two parts: one part containing 15 independent culture vials was built within a CO2 incubator (Figure 1), the other part, outside the incubator, contained the suction pumps and controlling equipment (Figure 2).\n\n(1) the magnetic stirrer plate with 15 positions. Upon this, (2) the vial holder array with the optical detection system placed upon the magnetic stirrer plate. (3) morbidostat culture vials that are placed and connected with silicone tubes going through a hole (4) at the back of the incubator to the peristaltic pumps outside the incubator.\n\nSilicone tubes connected to the peristaltic pumps, which are all placed against a plate fixed to the wall (2), (3) reservoirs bottles for plain media and media with antibiotic. (4) The 16-channel suction pump for waste.\n\nWe modified Toprak’s protocol13 and included one drug pump for each culture vial instead of two. This resulted in 30 peristaltic pumps: 15 medium pumps and 15 drug pumps for a capacity of 15 morbidostat culture vials. Consequently, we manually increased the drug concentrations by changing the drug reservoirs after a period of time, usually every three days. This modification made the morbidostat substantially cheaper.\n\nThe construction of the morbidostat proceeded in three steps. Firstly, the construction of the morbidostat culture vials. Secondly, the construction of the vial holder array and its optical detection system. These two parts were placed on a magnetic stirrer in an incubator, providing the growing conditions of N. gonorrhoeae: an atmosphere of 6% CO2 and a temperature of 36°C. Finally, each part was connected with silicone tubes and computer controlled peristaltic pumps for liquid transfer13.\n\n(i) Morbidostat culture vials. The morbidostat culture vial was a flat-bottom glass vial with a volume of 40 mL (Chemglass, CG-4902-08). It was closed at the top by an open-top GPI cap (Chemglass, CV-3750-0024) containing a Teflon insert with four holes (Euro-scientific). These holes were used for liquid injections (medium and antibiotics), waste extraction and for filtered air intake. Therefore, four pieces of PolyEtherEtherKetone (PEEK) tubing (BGB, 211609-25) were placed through the holes of the Teflon insert16. The PEEK tubing ends were connected to high temperature-resistant silicone tubing (Carl Roth, 9556.1) on the outside of the culture vial. Different parts of silicone tubing were connected using Luer connectors (male and female) (Nordson Medical, MTLL004-6005 and FTLL004-6005). Each culture vial contained a magnetic stirrer bar (Carl Roth, PK75.1), to maintain a constant stirring of the culture. All materials used for assembling, were temperature-resistant and autoclavable13.\n\n(ii) Vial holder array and its optical detection system. When the morbidostat was in operation mode, the morbidostat culture vials were placed in the holder array, a figure of the vial holder array with all its dimensions is shown in Figure 3. In the tube holder, infrared (IR) light emitting diodes (LEDs) (Velleman, L-7113E3BT) and photodetectors (Velleman, L-7113P3C) were machined in a black Delrin material by two openings, positioned at an angle of 135° to maximize scattered light detection. The LEDs and photodetectors were the optical detection system, and measured the optical density or turbidity in the morbidostat culture vials at set time intervals. The LEDs were connected to a relay-interface device, that could switch ON/OFF to a voltage of approximately 6V. The photo-detectors were connected with a data acquisition device (DAQ) card (Measurement Computing, USB-1616FS). This DAQ card recorded the analog voltage readings across the photo-detectors and sent a digital signal to the computer. The vial holder array sat on top of the 15-position magnetic stirrer (Carl Roth, EHY9.1) that ensured continuously stirring at a speed of 200 rotations per minute (rpm) in each culture vial13.\n\n(a) Top view of the vial holder array sitting on the magnetic stirrer plate, (b) Top view of the Delrin ring with the light emitting diode (LED) and detector, (c) Side view of the vial holder array which sits on the magnetic stirrer plate and (d) the dimensions of the LED/detector used.\n\n(iii) Assembling of the controlled peristaltic pump array. The last part of the construction was the connection of silicone tubes and computer controlled peristaltic pumps for liquid transfer to/from the morbidostat culture vials. All different parts with silicone tubes were connected to each other using Luer connectors. These silicone tubes exited the culture vials via the incubator (Memmert, ICO150med) through a hole at the backside. The 30 peristaltic pumps and the 16-channel suction pump were controlled with a relay interface device (Measurement Computing, USB-ERB08 and USB-ERB24), that is connected to a computer. The pumps were computer controlled using an algorithm coded in MATLAB software (MATLAB R2015b). The control software for the NG morbidostat is available from GitHub (see Software availability). The whole code was based on the code Toprak et al. used13. The code ‘ExperimentController.m’ was the main class file, which ran the whole experiment by controlling ‘PumpController.m’, ‘DataReceiver.m’ and ‘DataMonitor.m’ in parallel. ‘PumpController.m’ controlled the 30 peristaltic pumps and the 16-channel suction pump. The file ‘DataReceiver.m’ contained the code that read the voltage measurements by the photo-detectors. It converted the voltage readings automatically to turbidity measurements, depending on the calibration parameters. The file ‘DataMonitor.m’ monitored the real-time data to the user while the experiment was running. Algorithm parameters like dilution time, growth time and mixing time could be set for the experiment. The 16-channel peristaltic pump was used to remove the waste/excess volume in the morbidostat culture vials.\n\nTo make sure the whole system was sterile, all tubing was autoclaved at 121°C for 21 minutes followed by a washing cycle with bleach (7%), sterile water, ethanol (70%), sterile water and growth medium, respectively, before use13.\n\nWe used an inoculation volume of 10 µL from a N. gonorrhoeae bacterial suspension (WHO-F and WHO-X) of 4.0 McF in 12 mL GC Broth supplemented with 1% IsoVitaleX (BD BBL™) enrichment for each morbidostat culture vial. All culture vials were autoclaved at 121°C for 20 minutes before use. N. gonorrhoeae grew in the morbidostat in cycles of 21 minutes and after each cycle, depending on turbidity measurements and growth rate, an algorithm in the software diluted the suspension with 1 mL fresh medium or with 1 mL fresh medium containing antibiotics. We set 1.3 McF as a threshold for addition of fresh medium, to allow N. gonorrhoeae to adapt to the environment without being diluted. Fresh medium with antibiotic was injected when a threshold of 2.0 McF was exceeded and the net growth was positive, otherwise fresh medium was injected. The algorithm used, is shown in Figure 4. After 800 seconds, a 16-channel suction pump removed the excess liquid. We took samples every 2–3 days by disconnecting the culture vials and swiping an inoculating loop through the suspension. Samples were then grown on blood agar plates and after approximately 24 hours, we stored these cultures from the blood agar plates into skim milk at -80°C14. Azithromycin susceptibility testing was performed using E-tests (Biomerieux). In Figure 5 the influence of bacterial growth when adding antibiotic is shown.\n\n(a) Control algorithm used in the NG morbidostat. Thresholds used in this algorithm were based on values obtained from the (b) growth curve of N. gonorrhoeae, green and red arrows at the mid-log phase correspond with the thresholds for medium and antibiotic injections, respectively.\n\nRed arrows indicate where antibiotics were added to the culture. Every small peak in this curve corresponds to a cycle of 21 minutes.\n\n\nResults & discussion\n\nN. gonorrhoeae is very sensitive to environmental changes and requires a nutrient rich growth medium, we encountered a few issues during the optimization of our NG morbidostat.\n\ni. Temperature optimization. After the incubator was set and validated on a temperature of 36.5°C, we were not able to maintain proper growth at each position of the morbidostat. A particularly problematic position was the middle part of the vial holder. Temperature measurements using temperature probes inside the morbidostat culture vials, revealed temperatures ranging from 38°C to 41°C, which is too hot for optimal growth of N. gonorrhoeae. To solve this problem, we had to remove the upper plexi plate of the vial holder to maintain a homogenous and adequate temperature distribution between the culture vials. In addition, we changed the setpoint temperature in the incubator itself to 35°C, as the motor of the magnetic stirrer plate generates heat in its direct environment and N. gonorrhoeae is very sensitive to these environmental changes. With these adjustments, temperatures measured in the morbidostat culture vials were in the right temperature range (approximately 36°C) for all 15 vials.\n\nii. Contamination. Compared to previous morbidostat experiments described in literature11,13,15,17, where a minimal growth medium is used, N. gonorrhoeae requires a nutrient rich growth medium (GC Broth) which in its turn increases the risk of contamination.\n\nThe most common contaminant of our culture media were Bacillus species. This rapidly growing contaminant resulted in two major problems. First, the nutrients available in the medium were used by these contaminants thus insufficient for the gonococcus growing in the culture vials. Second, when we used 0.2 µm filters between (a) the media reservoir and the peristaltic pump and (b) the peristaltic pump and the morbidostat culture vial, filters were clogged very rapidly. As a consequence (a) the peristaltic pumps could no longer draw the medium into the system or (b) pressure overload occurred in the tubing between the peristaltic pump and the morbidostat culture vial, causing a disconnection between the peristaltic pumps and the tubing. Therefore, we do not recommend to work with these filters when there is high risk of contamination in the culture medium. Our next step was to limit contamination by placing the medium reservoir in a UV-light-box, but in less than 24 hours contamination reoccurred. Ultimately the only way we found to prevent this contamination was to add a vancomycin, colistin, nystatin and trimethoprim selective supplement (VCNT) to the growth medium. VCNT inhibits the growth of most other micro-organisms excluding N. gonorrhoeae18. This step solved the contamination problem.\n\nAs proof of concept, we used the NG morbidostat to generate resistance to azithromycin. In this pilot experiment we used single vials with two strains of N. gonorrhoeae (WHO-F and WHO-X) exposed to increasing concentrations of azithromycin. We used the same two strains of N. gonorrhoeae contemporaneously exposed to growth medium only, as controls.\n\nWe observed the highest bactericidal rate soon after the first azithromycin exposure. After this initial exposure, the population needed a longer time to return to their log phase than later on in the experiment. Figure 6 shows a growth curve of the population during the experiment.\n\nThe initial minimal inhibitory concentrations (MICs) of WHO-F and WHO-X were 0.125 µg/mL and 0.25 µg/mL respectively. In the first week, the MICs of WHO-F and WHO-X increased approximately 24-fold and 48-fold, respectively. By the end of the experiment (30 days), the MICs of WHO-F and WHO-X had increased more than 750-fold and 1000-fold, respectively (Table 1; Figure 7).\n\n\nConclusion\n\nIn conclusion, we described how to adapt the morbidostat device developed by Toprak et al.13 to a version that is able to maintain N. gonorrhoeae growth under constant antibiotic pressure. The key adaptations involved building the morbidostat within a CO2 incubator, fine-tuning the positioning of the vial holder array and the use of VCNT. This enabled us to induce high level azithromycin resistance in N. gonorrhoeae within 30 days. We plan to repeat these experiments for azithromycin and ceftriaxone in triplicate and perform whole-genome sequencing on those samples to characterize the sequential mutation steps in the resistance genesis. In future experiments we also plan to use the NG Morbidostat to evaluate how different antimicrobial combinations (including antiseptic products19 and bacteriophages) may be used to prevent the emergence of antimicrobial resistance in N. gonorrhoeae.\n\n\nData availability\n\nFigshare: data_proof_of_concept. https://dx.doi.org/10.6084/m9.figshare.798716920\n\nThis project contains the following underlying data:\n\ndata_proof_of_concept_experiment.xlsx (Proof of concept – in vitro evolution of resistance to azithromycin underlying data)\n\nraw_data_figure5.xlsx (Data underlying Figure 5)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nSoftware availability\n\nSource code: https://github.com/everhoeven/NG_Morbidostat\n\nArchived source code: http://doi.org/10.5281/zenodo.264343721\n\nLicence: MIT", "appendix": "Grant information\n\nThe project has been funded by the Institute of Tropical Medicine [pump prime 757224].\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nAuthors thank Ivo Jansegers (Institute of Tropical Medicine) for his technical help with all electronic parts and also Richard Neher for sharing information about the LED and pump components.\n\n\nReferences\n\nHolmes KK, Sparling PF, Stamm WE, et al.: Sexually Transmitted Diseases, Fourth Edition. McGraw Hill Professional, 2007. Reference Source\n\nRice PA, Shafer WM, Ram S, et al.: Neisseria gonorrhoeae: Drug Resistance, Mouse Models, and Vaccine Development. Annu Rev Microbiol. 2017; 71(1): 665–686. PubMed Abstract \| Publisher Full Text\n\nNdowa F, Lusti-Narasimhan M: The threat of untreatable gonorrhoea: implications and consequences for reproductive and sexual morbidity. Reprod Health Matters. 2012; 20(40): 76–82. PubMed Abstract \| Publisher Full Text\n\nWHO: Report on global sexually transmitted infection surveillance, 2018. WHO Geneva, ISBN 978-92-4-156569-1, 2018. Reference Source\n\nPublic health England: Sexually transmitted infections and chlamydia screening in England, 2015. Health Protection Report. Public Health England, 2016. 2016; 10(22). Reference Source\n\nECDC: Annual Epidemiological Report: Gonorrhoea. ECDC, Stockholm, 2015. Reference Source\n\nUnemo M, Shafer WM: Antimicrobial resistance in Neisseria gonorrhoeae in the 21st century: past, evolution, and future. Clin Microbiol Rev. 2014; 27(3): 587–613. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBignell C, Unemo M, European STI Guidelines Editorial Board: 2012 European guideline on the diagnosis and treatment of gonorrhoea in adults. Int J STD AIDS. 2013; 24(2): 85–92. PubMed Abstract \| Publisher Full Text\n\nWHO: WHO \| WHO guidelines for the treatment of Neisseria gonorrhoeae. WHO. [Online] [Accessed: 31-May-2018]. Reference Source\n\nWi T, Lahra MM, Ndowa F, et al.: Antimicrobial resistance in Neisseria gonorrhoeae: Global surveillance and a call for international collaborative action. PLoS Med. 2017; 14(7): e1002344. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEyre DW, Sanderson ND, Lord E, et al.: Gonorrhoea treatment failure caused by a Neisseria gonorrhoeae strain with combined ceftriaxone and high-level azithromycin resistance, England, February 2018. Euro Surveill. 2018; 23(27): 1800323. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatel AL, Chaudhry U, Sachdev D, et al.: An insight into the drug resistance profile & mechanism of drug resistance in Neisseria gonorrhoeae. Indian J Med Res. 2011; 134(4): 419–431. PubMed Abstract \| Free Full Text\n\nToprak E, Veres A, Yildiz S, et al.: Building a morbidostat: an automated continuous-culture device for studying bacterial drug resistance under dynamically sustained drug inhibition. Nat Protoc. 2013; 8(3): 555–567. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nToprak E, Veres A, Michel JB, et al.: Evolutionary paths to antibiotic resistance under dynamically sustained drug selection. Nat Genet. 2011; 44(1): 101–105. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDößelmann B, Willmann M, Steglich M, et al.: Rapid and Consistent Evolution of Colistin Resistance in Extensively Drug-Resistant Pseudomonas aeruginosa during Morbidostat Culture. Antimicrob Agents Chemother. 2017; 61(9): pii: e00043-17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPanayotov IV, Orti V, Cuisinier F, et al.: Polyetheretherketone (PEEK) for medical applications. J Mater Sci Mater Med. 2016; 27(7): 118. PubMed Abstract \| Publisher Full Text\n\nLiu PC, Lee YT, Wang CY, et al.: Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection. J Vis Exp. 2016; (115): e54426. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPace PJ, Catlin BW: Characteristics of Neisseria gonorrhoeae strains isolated on selective and nonselective media. Sex Transm Dis. 1986; 13(1): 29–39. PubMed Abstract \| Publisher Full Text\n\nChow EPF, Walker S, Hocking JS, et al.: A multicentre double-blind randomised controlled trial evaluating the efficacy of daily use of antibacterial mouthwash against oropharyngeal gonorrhoea among men who have sex with men: the OMEGA (Oral Mouthwash use to Eradicate GonorrhoeA) study protocol. BMC Infect Dis. 2017; 17(1): 456. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVerhoeven E: data_proof_of_concept. figshare. Fileset. 2019. http://www.doi.org/10.6084/m9.figshare.7987169.v2\n\neverhoeven: everhoeven/NG_Morbidostat: v2.0.0 (Version v2.0.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.2643437" }	[ { "id": "50218", "date": "25 Jun 2019", "name": "William M. Shafer", "expertise": [ "Reviewer Expertise My area of research deals with antibiotic resistance and Neisseria gonorrhoeae." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript is an important contribution to the field of antibiotic resistance development in Neisseria gonorrhoeae. The \"NG Morbidostat\" detailed by the authors has many advantages for studies on evolution of antibiotic resistance in culture. The construction and use of the system is well described. I have a few technical questions that require the attention of the authors:\nPlease indicate the colony type of the two strains employed (state of piliation and opacity).\n\nFor azithromycin, Etest strips can give variable results so it is important to verify the deduced MIC by the more accepted agar dilution method. Also, was the Etest assay performed on outgrowth of single colonies or the batch culture? Did the authors check single colonies?\n\nIt is not clear as to why blood agar plates were employed as opposed to the more conventional GC agar?\n\nIt would seem that the progression in azithromycin MICs represented initial mutations in the mtr locus followed by 23S rRNA genes, This could be checked quickly by PCR/sequencing.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "4921", "date": "16 Sep 2019", "name": "Jolein Laumen", "role": "Author Response", "response": "We would like to thank the reviewers for their valuable comments. Please find our responses below: 1.Please indicate the colony type of the two strains employed (state of piliation and opacity).This article describes the construction and optimization of the NG morbidostat. Its primary goal is to share how we constructed/optimized the morbidostat, and in particular how we overcame stumbling-blocks along the way. We thought this would be a useful contribution to the field as it may help other groups who may wish to construct in-vitro models to test various theories about gonococcal AMR selection. The article also includes a proof of concept experiment. We have recently completed larger scale experiments where we have conducted the various steps suggested by the reviewers such as describing the colony type, confirming MIC results with agar dilution and performed whole genome sequencing of the isolates every 3 to 5 days. These results will be described in a forthcoming paper. 2.For azithromycin, Etest strips can give variable results so it is important to verify the deduced MIC by the more accepted agar dilution method. Also, was the Etest assay performed on outgrowth of single colonies or the batch culture? Did the authors check single colonies?Because this was a proof of concept experiment, we performed E-tests on the batch culture. We have previously compared the results of azithromycin E-tests with agar dilution in our laboratory and found them to be comparable, as was also found by others (Liu H, Taylor TH, Pettus K, Trees D. Assessment of Etest as an alternative to agar dilution for antimicrobial susceptibility testing of Neisseria gonorrhoeae. Journal of clinical microbiology. 2014 May 1;52(5):1435-40). 3.It is not clear as to why blood agar plates were employed as opposed to the more conventional GC agar? We used blood agar plates instead of GC agar plates because this allowed us to check for a broader spectrum of contaminants. 4.It would seem that the progression in azithromycin MICs represented initial mutations in the mtr locus followed by 23S rRNA genes. This could be checked quickly by PCR/sequencing.The progression in azithromycin MIC indeed suggests initial mutations in the mtr locus followed by 23S rRNA genes. We are currently analyzing the whole genome sequencing data from our larger follow-up experiment and will then be able to provide this information in a subsequent paper." } ] }, { "id": "50219", "date": "01 Aug 2019", "name": "Olusegun O. Soge", "expertise": [ "Reviewer Expertise Antimicrobial resistance in Neisseria gonorrhoeae" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis interesting manuscript describes the construction and optimization of the ‘NG Morbidostat’ for in vitro selection of antimicrobial resistance in Neisseria gonorrhoeae. The NG Morbidostat would be valuable for real-time investigation of the evolution of antimicrobial resistance in N. gonorrhoeae. I have a few comments for the authors to consider.\nWhat is the rationale for selecting the two WHO strains used for the proof of concept experiment?\n\nDid the authors perform species confirmatory tests on the presumptive N. gonorrhoeae colonies cultured over the period of the experiment (30 days)?\n\nPlease mention the media and QC strains used for Etest.\n\nI would recommend performing agar dilution antimicrobial susceptibility testing with a standard panel of antimicrobials to confirm that there was no cross-resistance to other antimicrobials.\n\nIt has been described previously that high-level azithromycin resistance can develop rapidly in the laboratory in N. gonorrhoeae isolates with a single mutated 23S rRNA allele.1 What are the genetic and phenotypic characteristics of the two WHO strains? Could the presence of multiple resistance-conferring mutations, including a single-base-pair deletion within a 13-bp inverted-repeat sequence in the mtrR promoter of WHO X2 have contributed to the higher azithromycin MICs obtained when compared to WHO F?\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "4922", "date": "16 Sep 2019", "name": "Jolein Laumen", "role": "Author Response", "response": "We would like to thank the reviewers for their valuable comments. Please find our responses below: 1.What is the rationale for selecting the two WHO strains used for the proof of concept experiment? For the proof of concept experiment we chose WHO-F because this strain is susceptible to the major relevant anti-gonococcal antibiotics (penicillin G, tetracycline, ciprofloxacin, azithromycin, cefixime, ceftriaxone and spectinomycin)1. It thus enables us to study the evolution of AMR from a wildtype strain. The WHO-X strain was chosen because it is already resistant to multiple antibiotics, including penicillin G, tetracycline, ciprofloxacin, ceftriaxone (MIC 2) and cefixime (MIC 4)1. WHO-X also has the 13 bp inverted repeat in its mtrR promoter and has a slightly elevated azithromycin MIC (0.25). These features may enhance the development of resistance to azithromycin faster or via a different pathway. 2.Did the authors perform species confirmatory tests on the presumptive N. gonorrhoeae colonies cultured over the period of the experiment (30 days)? The presumptive N. gonorrhoeaecolonies of the proof of concept experiment were not confirmed by additional testing. However, in a soon to be published detailed follow-up experiment we performed whole genome sequencing of the obtained samples that confirmed the identity of the isolates as N. gonorrhoeae. 3.Please mention the media and QC strains used for Etest. E-tests were performed on GC-agar plates and ATCC strain 49226 and strain WHO-X were used for quality control. 4.I would recommend performing agar dilution antimicrobial susceptibility testing with a standard panel of antimicrobials to confirm that there was no cross-resistance to other antimicrobials. Thank you for this useful comment. We have just finished a more detailed follow-up experiment wherein we will report the antimicrobial susceptibility of different antimicrobials (tested via agar dilution). 5.It has been described previously that high-level azithromycin resistance can develop rapidly in the laboratory in N. gonorrhoeae isolates with a single mutated 23S rRNA allele.1What are the genetic and phenotypic characteristics of the two WHO strains? Could the presence of multiple resistance-conferring mutations, including a single-base-pair deletion within a 13-bp inverted-repeat sequence in the mtrR promoter of WHO X2 have contributed to the higher azithromycin MICs obtained when compared to WHO F? The WHO-F strain has a wild type genetic profile whereas the WHO-X strain contains various mutants, including the 13-bp inverted repeat sequence in the mtrRpromoter. The WHO-X strain does not have any mutations in the 23S rRNA gene. In our more detailed follow-up experiment we saw that the WHO-F strain also reached a MIC of >256 (determined by E-test). However, as in the current paper, the WHO-X strain developed high level resistance faster than WHO-F. This faster acquisition could be due to the genetic profile of WHO-X. Whole genome sequence analysis of this follow-up experiment will be described in a subsequent paper. References Unemo M, Golparian D, Sánchez-Busó L, Grad Y, Jacobsson S, Ohnishi M, Lahra MM, Limnios A, Sikora AE, Wi T, Harris SR. The novel 2016 WHO Neisseria gonorrhoeae reference strains for global quality assurance of laboratory investigations: phenotypic, genetic and reference genome characterization. Journal of Antimicrobial Chemotherapy. 2016 Jul 17;71(11):3096-108." } ] } ]	1	https://f1000research.com/articles/8-560
https://f1000research.com/articles/8-1194/v1	26 Jul 19	{ "type": "Research Article", "title": "Next-generation sequencing of microbial cell-free DNA for rapid noninvasive diagnosis of infectious diseases in immunocompromised hosts", "authors": [ "Jose F. Camargo", "Asim A. Ahmed", "Martin S. Lindner", "Michele I. Morris", "Shweta Anjan", "Anthony D. Anderson", "Clara E. Prado", "Sudeb C. Dalai", "Octavio V. Martinez", "Krishna V. Komanduri", "Asim A. Ahmed", "Martin S. Lindner", "Michele I. Morris", "Shweta Anjan", "Anthony D. Anderson", "Clara E. Prado", "Sudeb C. Dalai", "Octavio V. Martinez", "Krishna V. Komanduri" ], "abstract": "Background: Cell-free DNA (cfDNA) sequencing has emerged as an effective laboratory method for rapid and noninvasive diagnosis in prenatal screening testing, organ transplant rejection screening, and oncology liquid biopsies. Methods: Here we report our experience using next-generation sequencing (NGS) for detection of microbial cfDNA in a cohort of ten immunocompromised patients with febrile neutropenia or deep-seated infection. Results: Among five hematological malignancy patients, for whom a microbiological diagnosis was established, pathogen identification by cfDNA NGS demonstrated 100% positive agreement with conventional diagnostic laboratory methods. Further, cfDNA identified the etiological agent in two patients with culture negative sepsis who had undergone hematopoietic stem cell transplant. Conclusion: These data support the clinical utility of measurement of microbial cfDNA sequencing from peripheral blood for rapid noninvasive diagnosis of infections in immunocompromised hosts. Larger studies are needed.", "keywords": [ "Cell-free microbial DNA", "next generation sequencing", "infection", "immunocompromised host", "hematopoietic stem cell transplant" ], "content": "Introduction\n\nInfections are a leading cause of morbidity and mortality among immunocompromised individuals1–4. Bacteremia occurs in up to 25% of all patients with neutropenia and fever5. Infection is a leading cause of non-relapse mortality among hematopoietic cell transplantation (HCT) recipients6. The incidence of bacteremia7–9 and double-stranded DNA viral reactivation10 is higher than 40% and 90%, respectively, within the first 100 days post-transplant. The cumulative incidence rates of proven/probable invasive fungal infections during the first year after allogeneic HCT with non-myeloablative conditioning is 19%11. Infection is also a common complication of chimeric antigen receptor-modified T (CAR-T)-cell immunotherapy with 28-day cumulative incidence of 23% after CAR-T-cell infusion12.\n\nEstablishing a microbiological diagnosis of infectious diseases in this vulnerable population is often challenging for a number of reasons. i) Prior exposure to antibiotics and antifungals which confounds the yield of blood cultures; indeed, most patients with neutropenia and fever will have no infectious etiology documented5. ii) Low sensitivity of mycobacterial and fungal cultures; some microorganisms, such as fastidious bacteria, mycobacteria and dimorphic fungi require longer incubation periods; and blood cultures in almost half of patients with candidemia are negative13,14. iii) Tissue biopsies are often precluded due to the risk of bleeding in the setting of thrombocytopenia, coagulopathy in those with liver disease or hemodynamic instability in critically ill patients. A delay in diagnosis in patients with invasive fungal infection results in higher mortality15,16. Thus, there is an unmet need for novel, rapid, cost-effective, noninvasive diagnostic methods in the field.\n\nCell-free DNA (cfDNA) technology has been used successfully in noninvasive prenatal testing, organ transplant rejection screening, and oncology liquid biopsies17–22. In recent years, this technology has been developed for use in infectious disease diagnostics23,24. Detection of microbial cfDNA by next generation sequencing (NGS) is an accurate and precise way of identifying and quantifying pathogens25. The Karius® Test relies on sequencing of microbial cfDNA circulating in plasma to identify over 1,000 pathogens, including bacteria, viruses and fungi, from a 5 ml blood sample25. This novel diagnostic tool has been recently validated in a study showing that microbial cfDNA NGS identified 94% of microbes identified by conventional blood culture in patients with sepsis25 and has excellent correlation with quantitative PCR testing in patients with cytomegalovirus (CMV)23,25.\n\nRecent reports indicate that NGS measuring microbial cfDNA is useful in the diagnosis of cases of Streptococcus pneumoniae-related hemolytic uremic syndrome, Coxiella burnetii endocarditis, invasive Mycobacterium chimaera infection, Nocardia cyriacigeorgica pneumonia, Capnocytophaga canimorsus sepsis, M. tuberculosis complex and M. haemophilum infections, M. bovis aortitis; Candida spp., Aspergillus spp., non-Aspergillus molds invasive infections; Pneumocystis jirovecii pneumonia (PJP), Toxoplasma gondii infection and chorioamnionitis, among others24,26–33. Among 21 patients with culture-positive infective endocarditis, cfDNA NGS identified the same organism as blood cultures in 20 patients (95% sensitivity) and additionally identified Enterococcus faecalis in one out of the three patients with definitive culture-negative endocarditis34. Of note, in this study the cfDNA NGS test identified pathogens causing endocarditis in patients pre-treated with antibiotics up to 30 days prior to initial sample collection.\n\nHere we evaluated the clinical utility of NGS for detection of microbial cfDNA in plasma in a cohort of ten patients receiving chemotherapy or transplants with episodes of febrile neutropenia, sepsis or documented infection.\n\n\nMethods\n\nAdult patients followed at the Sylvester Comprehensive Cancer Center were enrolled between July 31 and October 2, 2018. Inclusion criteria were: i) age >18 years old; ii) patients must have received chemotherapy or transplant; and iii) must have had a febrile illness or documented infection (e.g., positive blood cultures, clinical/radiographic evidence of pneumonia). There were no exclusion criteria. In this proof-of-concept study, most of the patients had an established diagnosis of infection prior to NGS testing. The study was approved by the University of Miami Institutional Review Board (IRB approval #20080899), consistent with principles in the Declaration of Helsinki. Each participant provided written informed consent for their inclusion in the study. No sample size calculation was done; instead the number of patients enrolled was entirely dependent on the number of cfDNA kits made available for the pilot study.\n\nBlood samples (5 mL) were collected in BD vacutainer plasma preparation tubes. Samples were collected at the time of suspected or confirmed infection diagnosis. Within 1 hour of sample collection, tubes were spun down at 1,100 RCF for 10 min at room temperature. Samples were shipped overnight to Karius, Inc. (Redwood City, CA).\n\nCell-free DNA was extracted from plasma, NGS libraries were prepared, and sequencing was performed on an Illumina NextSeq®500. Sequencing reads identified as human were removed, and remaining sequences were aligned to a curated pathogen database. Any of over 1,000 organisms in the Karius clinical reportable range found to be present above a predefined statistical threshold were reported as previously described24. The quantity for each organism identified was expressed in Molecules Per Microliter (MPM), the number of DNA sequencing reads from the reported organism present per microliter of plasma.\n\nReference database and QC. Reference genomes for Homo sapiens and microorganisms (bacteria, viruses, fungi/molds, and other eukaryotic pathogens) were retrieved from the National Center for Biotechnology Information (NCBI) ftp site (NCBI, U.S. National Library of Medicine (NLM), Human Genome, release GRCh38.p7, and NCBI, U.S. NLM, Microbial Genomes, respectively). Sequence similarities between microorganism references were inspected to identify taxonomic mislabeling and sequence contamination. From the reference genomes passing these quality controls, a subset was selected to maximize sequence diversity. As part of the selection process, NCBI BioSample data were used to ensure the inclusion of reference genomes from both clinical and non-clinical isolates. The final reference genome dataset included over 21,000 reference genomes, containing over 2.7 million sequences. Selected sequences were collected into a single FASTA file and used to generate our microorganism reference BLAST database. A subset of these taxa, including 1251 clinically significant microorganisms, was used as the clinical reportable range.\n\nClinical reportable range (CRR). The selection of organisms in our clinical reportable range (CRR) was performed as follows. A candidate list was generated by two board-certified infectious disease physicians by including (a) DNA viruses, culturable bacteria, additional fastidious and unculturable bacteria, mycobacteria, and eukaryotic pathogens from the standard text35 and a number of infectious disease references, (b) organisms in the pathogen database referenced in published case reports, and (c) reference genomes sequenced from human clinical isolates (as indicated by the NCBI BioSample resource) with publications supporting pathogenicity. Organisms from the above list that were associated with high-quality reference genomes, as determined by our reference database QC process (see above), were used to further narrow the range. Finally, organisms at risk of generating common false-positive calls because of sporadic environmental contamination were removed. The sequence database is continuously curated to minimize human cross-reactivity as well as cross-reactivity between pathogens and is screened to mitigate contamination with sequences from human or other organisms.\n\nSequencing. Plasma samples were thawed, centrifuged at 16,000 RCF for 10 min, and spiked with a known concentration of synthetic DNA molecules for quality control purposes. Cell-free DNA was extracted from 0.5 mL plasma using a magnetic bead-based method (Omega Bio-tek Mag-Bind® cfDNA kit; catalog number M3298-01, Norcross, GA). DNA libraries for sequencing are constructed using a modified Ovation® Ultralow System V2 library preparation kit (NuGEN, San Carlos, CA). Negative controls (buffer only instead of plasma) and positive controls (healthy plasma spiked with a known mixture of microbial DNA fragments) were processed alongside patient samples in every batch. Samples were multiplexed with other samples and sequenced on an Illumina NextSeq® 500.\n\nAnalysis pipeline. Primary sequencing output files were processed using bcl2fastq (v2.17.1.14) to generate the demultiplexed sequencing reads files. Reads were filtered based on sequencing quality and trimmed based on partial or full adapter sequence. The bowtie2 (version 2.2.4) tool was used to align the remaining reads against Karius' human and synthetic-molecules references. Sequencing reads exhibiting strong alignment against the human references or the synthetic molecule references were collected and excluded from further analysis. Remaining reads were aligned against Karius' proprietary microorganism reference database using NCBI-blast (version 2.2.30+). A mixture model was used to assign a likelihood to the complete collection of sequencing reads that included the read sequence probabilities and the (unknown) abundances of each taxon in the sample. An expectation-maximization algorithm was applied to compute the maximum likelihood estimate of each taxon abundance. Only taxa whose abundances rejected the null hypothesis of originating from environmental contamination (as calculated from the negative controls) at high significance levels were reported. The quantity for each organism identified was expressed in molecules per microliter (MPM), the number of DNA sequencing reads from the reported organism present per microliter of plasma. The entire process from DNA extraction through analysis was typically completed within 28 hours.\n\n\nResults\n\nThe characteristics of the patients studied are presented in Table 1. The median age was 56 years (range, 20–65) with 60% of participants males. Except for a kidney transplant recipient, all other patients had underlying hematological malignancy and/or had received an HCT. All but one (patient #2) were admitted in the hospital at the time of clinical evaluation. All the patients were receiving antimicrobials at the time of plasma sample collection. Three patients had neutropenia (absolute neutrophil count <500/µL) at the time of febrile illness. All febrile patients had blood cultures collected within 24 hours of plasma sample collection for NGS.\n\na Refers to empiric or targeted therapy only. It does not include days of antimicrobial prophylaxis.\n\nb Blood cultures were obtained within 24h of plasma sample for NGS in all patients and resulted as negative unless specified otherwise in the table.\n\nc Reference value is the 97.5th percentile in self-reported healthy adults for whom the Karius® Test was performed\n\nd Correlation between Karius® Test and standard laboratory methods\n\neAspergillus fumigatus reads were present in the raw data but below the threshold for a positive test result.\n\nf Initial cfDNA testing performed 7 weeks prior had only identified S. epidermidis and EBV. At that time, BAL and transbronchial biopsy results were unrevealing.\n\nALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; BAL, bronchoalveolar lavage; CAR-T, chimeric antigen receptor-modified T-cell immunotherapy; cfDNA, cell-free DNA; CMV, cytomegalovirus; CVC, central venous catheter; DLBCL, diffuse large B cell lymphoma; GI-GVHD, gastrointestinal graft-versus-host disease; HCT, hematopoietic cell transplantation; F, female; M, male; MPM, molecules per microliter; NGS, next-generation sequencing; NLH, Non-Hodgkin lymphoma; SOB, shortness of breath.\n\nThe kidney transplant recipient had an Aspergillus fumigatus deep-seated abdominal abscess, and Aspergillus cfDNA levels, although detected in plasma, were below the positive reporting threshold. However, among patients with hematological malignancy in whom a microbiological diagnosis was established (n=5), cfDNA NGS testing correlated with other methods in all cases (100% sensitivity). This included patients with proven/probable invasive aspergillosis, PJP, Stenotrophomonas maltophilia bacteremia, CMV and adenovirus viremia. Among four patients with hematological malignancy with negative standard laboratory testing, the NGS test identified causes of bacterial sepsis in two patients (Table 1), both of whom had a compatible clinical scenario and experienced good clinical response to antibiotic therapy with resolution of fever and hypotension.\n\n\nDiscussion\n\nHere we report our experience using cfDNA NGS in the evaluation of immunocompromised patients—predominantly those with hematological malignancy—with febrile illness or documented invasive infections. The study cohort included a heterogeneous group of clinical scenarios, including deep-seated pyogenic abdominal infection, pulmonary nodules/pneumonia, neutropenic fever, and septic shock. The results of this proof-of-concept study, where most of the patients had an established diagnosis of infection prior to NGS testing, complement recent reports studying the use of cfDNA NGS in immunocompromised hosts. In a recent study of 55 patients with neutropenic fever, cfDNA testing had positive agreement with conventional blood cultures in 9 of 10 patients in whom blood cultures identified a causative organism of sepsis. Using clinical adjudication by three infectious diseases specialists, cfDNA NGS had a sensitivity of 85.4% (41/48) and specificity of 100% (7/7)36. Thus, this test is a promising diagnostic tool in neutropenic fever, a clinical scenario where conventional work up fails to identify an etiological agent in a majority of cases5. Another study evaluated 40 patients with prolonged neutropenia and fever (>96h) despite administration of antibiotics for suspected fungal infection (the authors excluded patients who had received antifungal therapy for >3 days); in this study cfDNA NGS identified fungal pathogens including Aspergillus fumigatus, Rhizopus spp., Candida albicans, Candida glabrata and Pneumocystis jirovecii37.\n\nIn a recent report by Hong et al.24, in seven out of nine subjects (including seven immunocompromised hosts) with proven deep-seated invasive fungal infection, plasma NGS testing detected the same fungus identified from the biopsy tissue at the genus level. The fungi identified by plasma NGS included Aspergillus spp. and non-Aspergillus molds such as Scedosporium, Rhizopus, and Cunninghamella24. In that report, there was one case where the plasma sample was obtained after at least 15 days of anti-Aspergillus therapy, and NGS testing did not identify the causal organism of invasive fungal infection. Similarly, for the kidney transplant patient reported here with invasive aspergillosis, in whom Aspergillus fumigatus cfDNA levels in plasma were detected below the reporting threshold, several months of antifungal therapy had been administered prior to the time of plasma collection. Thus, prolonged antifungal therapy prior to sample collection (e.g., >7-14 days) might interfere with detection of fungal DNA.\n\nAlthough NGS has been used for screening of allograft rejection in solid organ transplant recipients17–19,23, there is limited data with the use of NGS for diagnosis of infections in this population. A recent study demonstrated strong correlation between clinical test results and cfDNA derived from CMV in a cohort of lung transplant recipients23. In addition, cfDNA revealed undiagnosed cases of infection with microsporidia and pathogenic viruses, including adenovirus and human herpesvirus 6 among lung transplant patients23.\n\nRecently, Fung et al. reported three patients who received allogeneic HCT transplant in whom NGS cfDNA facilitated the diagnosis of an uncommon presentation of Chlamydia trachomatis and recurrent and metastatic complications of Staphylococcus aureus bacteremia before standard microbiology38.\n\nThe fact that in our cohort cfDNA NGS testing identified the cause of febrile illness in two patients with culture-negative sepsis who had a compatible clinical syndrome and responded well to antibiotic therapy supports the notion that NGS testing can be a useful diagnostic tool, particularly when conventional blood cultures are negative. The Karius® Test pathogen-specific reference ranges have been established using cfDNA levels from healthy donors. Patient #8 had detectable levels of Torque teno virus, which belongs to Anelloviridae family and is considered to lack pathogenic potential; this suggests the possibility that cfDNA NSG might on occasion yield detection of members of the commensal microbiota or viroma. To our surprise, however, even though many of the patients tested had mucosal barrier damage (e.g., mucositis) allowing for bacterial translocation from the gut, the Karius® Test did not show a non-specific gut flora signal. The test was negative in patients in whom we failed to establish a microbiological diagnosis for their febrile illness, and when positive, typically correlated with conventional laboratory testing. Whether the currently defined cfDNA thresholds are optimal for identifying and quantifying pathogens of clinical relevance in highly vulnerable immunocompromised hosts will require further study. Importantly, the turnaround time for results was consistently within 48 hours, which is quite rapid considering that samples were shipped overnight from our institution located in Florida to the Karius Inc. laboratory in California.\n\nLack of control group, small number of patients and the heterogeneity of the cohort in terms of underlying diseases and causes of immunosuppression represent major limitations of this report. Larger clinical trials evaluating plasma NGS in patients with cancer and undergoing transplant are ongoing (NCT03226158, NCT03262584, NCT02912117, NCT02804464). Until larger cohort data becomes available, our observations suggest that detection of microbial cfDNA using NGS is valuable for the rapid noninvasive diagnosis of infectious complications following chemotherapy or transplantation.\n\n\nConclusion\n\nOur data, along with a number of recent reports, support the clinical utility of the measurement of microbial cfDNA in peripheral blood using NGS for rapid noninvasive diagnosis of infections in immunocompromised hosts. As with other novel laboratory diagnostics used in clinical practice, the results of cfDNA NGS technology need to be interpreted with caution and in conjunction with other laboratory, radiological and clinical findings. Larger studies are needed to validate these findings.\n\n\nData availability\n\nMicrobial cfDNA NGS for Rapid Noninvasive Diagnosis of Infectious Diseases in Immunocompromised Hosts, BioProject accession number PRJNA55427", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work\n\n\nAcknowledgments\n\nThis work was supported by Karius, Inc., Redwood City, CA.\n\n\nReferences\n\nTomblyn M, Chiller T, Einsele H, et al.: Guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective. Biol Blood Marrow Transplant. 2009; 15(10): 1143–1238. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFishman JA: Infection in solid-organ transplant recipients. N Engl J Med. 2007; 357(25): 2601–2614. PubMed Abstract \| Publisher Full Text\n\nSyrjala H, Ohtonen P, Kinnunen U, et al.: Blood stream infections during chemotherapy-induced neutropenia in adult patients with acute myeloid leukemia: treatment cycle matters. Eur J Clin Microbiol Infect Dis. 2010; 29(10): 1211–1218. PubMed Abstract \| Publisher Full Text\n\nBallen K, Woo Ahn K, Chen M, et al.: Infection Rates among Acute Leukemia Patients Receiving Alternative Donor Hematopoietic Cell Transplantation. Biol Blood Marrow Transplant. 2016; 22(9): 1636–1645. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFreifeld AG, Bow EJ, Sepkowitz KA, et al.: Clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the infectious diseases society of america. Clin Infect Dis. 2011; 52(4): e56–93. PubMed Abstract \| Publisher Full Text\n\nYoo JH, Lee DG, Choi SM, et al.: Infectious complications and outcomes after allogeneic hematopoietic stem cell transplantation in Korea. Bone Marrow Transplant. 2004; 34(6): 497–504. PubMed Abstract \| Publisher Full Text\n\nBock AM, Cao Q, Ferrieri P, et al.: Bacteremia in blood or marrow transplantation patients: clinical risk factors for infection and emerging antibiotic resistance. Biol Blood Marrow Transplant. 2013; 19(1): 102–108. PubMed Abstract \| Publisher Full Text\n\nKikuchi M, Akahoshi Y, Nakano H, et al.: Risk factors for pre- and post-engraftment bloodstream infections after allogeneic hematopoietic stem cell transplantation. Transpl Infect Dis. 2015; 17(1): 56–65. PubMed Abstract \| Publisher Full Text\n\nGudiol C, Garcia-Vidal C, Arnan M, et al.: Etiology, clinical features and outcomes of pre-engraftment and post-engraftment bloodstream infection in hematopoietic SCT recipients. Bone Marrow Transplant. 2014; 49(6): 824–830. PubMed Abstract \| Publisher Full Text\n\nHill JA, Mayer BT, Xie H, et al.: The cumulative burden of double-stranded DNA virus detection after allogeneic HCT is associated with increased mortality. Blood. 2017; 129(16): 2316–2325. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFukuda T, Boeckh M, Carter RA, et al.: Risks and outcomes of invasive fungal infections in recipients of allogeneic hematopoietic stem cell transplants after nonmyeloablative conditioning. Blood. 2003; 102(3): 827–833. PubMed Abstract \| Publisher Full Text\n\nHill JA, Li D, Hay KA, et al.: Infectious complications of CD19-targeted chimeric antigen receptor-modified T-cell immunotherapy. Blood. 2018; 131(1): 121–130. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClancy CJ, Nguyen MH: Finding the \"missing 50%\" of invasive candidiasis: how nonculture diagnostics will improve understanding of disease spectrum and transform patient care. Clin Infect Dis. 2013; 56(9): 1284–1292. PubMed Abstract \| Publisher Full Text\n\nMiller JM, Binnicker MJ, Campbell S, et al.: A Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2018 Update by the Infectious Diseases Society of America and the American Society for Microbiology. Clin Infect Dis. 2018; 67(6): 813–816. PubMed Abstract \| Publisher Full Text\n\nMorrell M, Fraser VJ, Kollef MH: Delaying the empiric treatment of candida bloodstream infection until positive blood culture results are obtained: a potential risk factor for hospital mortality. Antimicrob Agents Chemother. 2005; 49(9): 3640–3645. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvon Eiff M, Roos N, Schulten R, et al.: Pulmonary aspergillosis: early diagnosis improves survival. Respiration. 1995; 62(6): 341–347. PubMed Abstract \| Publisher Full Text\n\nBloom RD, Bromberg JS, Poggio ED, et al.: Cell-Free DNA and Active Rejection in Kidney Allografts. J Am Soc Nephrol. 2017; 28(7): 2221–2232. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchutz E, Fischer A, Beck J, et al.: Graft-derived cell-free DNA, a noninvasive early rejection and graft damage marker in liver transplantation: A prospective, observational, multicenter cohort study. PLoS Med. 2017; 14(4): e1002286. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDe Vlaminck I, Valantine HA, Snyder TM, et al.: Circulating cell-free DNA enables noninvasive diagnosis of heart transplant rejection. Sci Transl Med. 2014; 6(241): 241ra277. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAllyse MA, Wick MJ: Noninvasive Prenatal Genetic Screening Using Cell-free DNA. JAMA. 2018; 320(6): 591–592. PubMed Abstract \| Publisher Full Text\n\nRossi G, Mu Z, Rademaker AW, et al.: Cell-Free DNA and Circulating Tumor Cells: Comprehensive Liquid Biopsy Analysis in Advanced Breast Cancer. Clin Cancer Res. 2018; 24(3): 560–568. PubMed Abstract \| Publisher Full Text\n\nUlrich BC, Paweletz CP: Cell-Free DNA in Oncology: Gearing up for Clinic. Ann Lab Med. 2018; 38(1): 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDe Vlaminck I, Martin L, Kertesz M, et al.: Noninvasive monitoring of infection and rejection after lung transplantation. Proc Natl Acad Sci U S A. 2015; 112(43): 13336–13341. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHong DK, Blauwkamp TA, Kertesz M, et al.: Liquid biopsy for infectious diseases: sequencing of cell-free plasma to detect pathogen DNA in patients with invasive fungal disease. Diagn Microbiol Infect Dis. 2018; 92(3): 210–213. PubMed Abstract \| Publisher Full Text\n\nBlauwkamp TA, Thair SA, Rosen MJ, et al.: Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease. Nat Microbiol. 2019; 4(4): 663–674. PubMed Abstract \| Publisher Full Text\n\nYonts A, Farnaes L, Venkatasubrahmanyam S, et al.: Streptococcus pneumoniae-Related Hemolytic Uremic Syndrome (pHUS) and the Identification of Matched Cross-Country Strains by Next-Generation Sequencing (NGS). IDWeek. 2018.\n\nKondo MD, Dalai S, Westblade L, et al.: Diagnosis and Genotyping of Coxiella burnetii Causing Endocarditis in a Patient with Prosthetic Pulmonary Valve Replacement (PVR) Using Next-Generation Sequencing (NGS) of Plasma. Open Forum Infect Dis. aper presented at: IDWeek, 2018; 5(suppl_1): S11–S12. Publisher Full Text\n\nNayakwadi-Singer M, Johnston S, Kulhanjian J, et al.: Detection of Nocardia cyriacigeorgica from a Deep Pulmonary Infection Using a Novel Plasma-Based Next Generation Sequencing Assay. ASM Microbe. 2017.\n\nNomura J, Rieg G, Bluestone G, et al.: Rapid Detection of Invasive Mycobacterium chimaera Infection by Using a Novel Plasma-Based Next-Generation Sequencing Assay. Open Forum Infect Dis. 2017; 4(suppl_1): S174. Publisher Full Text\n\nStrand A, Hong DK, Fowler VG Jr, et al.: Diagnosis of Capnocytophaga canimorsus Sepsis by a Novel Cell-free DNA Sequencing Assay. ASM Microbe. 2016.\n\nHong DK, Truong C, N B: Diagnosis of Mycobacterium tuberculosis and Non-tuberculous Mycobacterium Infections Using a Novel Plasma-Based Next-Generation Sequencing Assay. ASM Microbe. 2016.\n\nCooper C, Hong D, Blair L, et al.: Diagnosis of BCG Aortitis by Plasma Metagenomic Sequencing. Open Forum Infect Dis. IDWeek; 2018; 5(suppl_1): S290. Publisher Full Text \| Free Full Text\n\nWitt RG, Blair L, Frascoli M, et al.: Detection of Pathogen Cell-Free DNA in Maternal Plasma in Patients with Chorioamnionitis Using a Non-Invasive Next-Generation Sequencing Assay. SRI 65th Annual Scientific Meeting. 2018.\n\nShah P, Ruffin F, Seng H, et al.: Direct Detection and Quantification of Bacterial Cell-free DNA in Patients with Infective Endocarditis (IE) Using the Karius Plasma Next Generation Sequencing (NGS) Test. Open Forum Infect Dis. IDWeek; 2018; 5(suppl_1): S12. Publisher Full Text\n\nBennett JE, Dolin R, Blaser MJ: Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases. (Saunders, Philadelphia, PA; 2014). Reference Source\n\nBenamu E, Gajurel K, Anderson JN, et al.: Evaluation of the Karius Plasma Next-Generation Sequencing Cell-free Pathogen DNA Test to Determine the Etiology of Infection and Impact on Anti-Microbial Management in Patients with Severe Neutropenia and Fever. Open Forum Infect Dis. IDWeek; 2018; 5(Suppl 1): S680. Publisher Full Text \| Free Full Text\n\nArmstrong A, Rossoff J, Aquino R, et al.: Plasma Next-Generation Sequencing for Pathogen Detection in Pediatric Patients at Risk for Invasive Fungal Infection. Open Forum Infect Dis. IDWeek; 2018; 5(suppl_1): S598. Publisher Full Text \| Free Full Text\n\nFung M, Zompi S, Seng H, et al.: Plasma Cell-Free DNA Next-Generation Sequencing to Diagnose and Monitor Infections in Allogeneic Hematopoietic Stem Cell Transplant Patients. Open Forum Infect Dis. 2018; 5(12): ofy301. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "52481", "date": "04 Sep 2019", "name": "William Muller", "expertise": [ "Reviewer Expertise Clinical pediatric infectious diseases with an emphasis on transplant ID", "antimicrobial clinical trials in pediatric patients" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCamargo et al. report on results of plasma next-generation sequencing (NGS) for infectious diagnosis in a series of ten patients at risk for infection due to underlying immunocompromise. In addition to demonstrating concordance of NGS testing with conventional microbiologic diagnostic testing methods in seven of the ten patients, NGS testing identified a possibly causative organism in two cases in which conventional test results were negative. In one of the ten patients, PCR and culture detected Aspergillus; while NGS was able to detect this organism, it was below the assay detection limit. The authors conclude that this pilot study supports the conclusion that there may be clinical benefit for using this test in this population of patients, warranting more rigorous studies of test performance.\n\nOverall, the manuscript does not contain significant flaws that would preclude indexing. The authors appropriately acknowledge the limitations of this small pilot study, which by design is not able to determine detailed test performance characteristics such as sensitivity or specificity. Accordingly, my comments are fairly minor and include:\nThe methods and results sections report the NGS data in “molecules per microliter” (MPM), which is defined as “the number of DNA sequencing reads from the reported organism present per microliter of plasma.” Although this is a fairly straightforward concept, in the context of sequencing it is not totally clear how the more commonly used concepts of sequencing “breadth” and depth” (e.g., as discussed by Sims et al., 20141) apply to MPM. Is it possible to explain MPM in a little more detail? E.g., is there a minimum depth of sequencing that needs to be satisfied for there to be \"one\" MPM? Does every base in a sequence need to have a certain number of reads?\n\nThe methods also refer to the removal of possible “false-positive calls” from common environmental contaminants – can any examples of organisms that would fit these criteria be provided? Since immunocompromised patients may be at risk of infection from uncommon organisms associated with their environment (particularly uncommon moulds), it is important to understand what may not be reported.\n\nIt’s not completely clear from the text whether results of NGS testing were used for clinical care. Although in most cases it is stated that an established diagnosis of infection was made prior to NGS testing, at least two patients had negative conventional testing and were reported to respond to therapy directed at the organisms identified by NGS. Were these patients responding to empiric treatment, or did NGS results direct the treatment?\n\nSeveral places in the manuscript refer to the abdominal infection in the kidney transplant patient as “deep-seated,” but only once is the more medically precise term “abscess” used to describe this infection. Also, can more details about this infection be provided? This is important as the patient appears to have had negative testing (unless the detection limit was lowered below that which the assay typically uses), providing additional clues to situations in which NGS testing may provide false negative results (more on this in the next comment).\n\nAll of the patients had received some antibiotic (antibacterial and/or antifungal) treatment at the time of NGS testing, and one of the more impressive and useful aspects of the test is that it may be able to detect organisms in the setting of effective treatment. However, although a duration of treatment is reported in Table 1, whether this treatment would have been effective against the organism identified is not clear, complicating the interpretation of the data. For example, the Discussion notes that “several months” of treatment had been given to the patient with the Aspergillus abscess prior to NGS testing, but it is not totally clear if it was all directed against Aspergillus – if so, the negative result is less concerning (and the ability to detect the organism below the reportable threshold is still impressive). Similarly, did patient 10 receive 129 days of antifungal treatment with anti-Aspergillus activity prior to his positive test? Although it is understandable why prophylactic treatment would not generally be reported for all patients, was patient 4 receiving any prophylaxis against Pneumocystis?\n\nThere are a handful of fairly minor editorial corrections also, including:\nOn page 4 the “clinically reportable range” is referred to as “our” clinically reportable range – although it is acknowledged that three of the authors are from the company which performed the NGS testing, given that certain details of the testing are ultimately proprietary perhaps a more generic statement (as simple as “the clinically reportable range”) would be preferred.\n\nIn that same paragraph, although the Mandell textbook is considered by many to be the “go-to” reference for clinical infectious diseases, perhaps it could be referred to as “a clinical infectious diseases reference textbook” or some less subjective phrase? Also, it appears the 8th edition is being referenced, which I believe is actually from 2015 (not 2014)?\n\nThe third paragraph in the left column on page 7 contains the phrase “there is limited data…” – as data is plural this should be corrected to “there are limited data…”.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4917", "date": "18 Sep 2019", "name": "Jose Camargo", "role": "Author Response", "response": "Reviewer’s comment: Camargo et al. report on results of plasma next-generation sequencing (NGS) for infectious diagnosis in a series of ten patients at risk for infection due to underlying immunocompromise. In addition to demonstrating concordance of NGS testing with conventional microbiologic diagnostic testing methods in seven of the ten patients, NGS testing identified a possibly causative organism in two cases in which conventional test results were negative. In one of the ten patients, PCR and culture detected Aspergillus; while NGS was able to detect this organism, it was below the assay detection limit. The authors conclude that this pilot study supports the conclusion that there may be clinical benefit for using this test in this population of patients, warranting more rigorous studies of test performance. Overall, the manuscript does not contain significant flaws that would preclude indexing. The authors appropriately acknowledge the limitations of this small pilot study, which by design is not able to determine detailed test performance characteristics such as sensitivity or specificity. Accordingly, my comments are fairly minor and include: Author’s response: Thank you for this thorough review of the manuscript and your insightful comments that have improved the clarity and quality of the paper. Below there is a point-by-point response your inquiries. Reviewer’s comment: The methods and results sections report the NGS data in “molecules per microliter” (MPM), which is defined as “the number of DNA sequencing reads from the reported organism present per microliter of plasma.” Although this is a fairly straightforward concept, in the context of sequencing it is not totally clear how the more commonly used concepts of sequencing “breadth” and depth” (e.g., as discussed by Sims et al., 20141) apply to MPM. Is it possible to explain MPM in a little more detail? E.g., is there a minimum depth of sequencing that needs to be satisfied for there to be \"one\" MPM? Does every base in a sequence need to have a certain number of reads? Author’s response: We revised the text to clarify the connection between MPM and sequencing depth. In the Karius Test, the total number of reads observed per organism is often orders of magnitude lower than in applications where the concepts of sequencing breadth and depth are typically used (such as genome assembly). There is indeed a minimum amount of sequencing information that must be obtained in order for a sample to pass quality control criteria which is proportional to the concentration of cell-free DNA in that patient’s sample. Importantly, the MPM value is not affected by sequencing depth or human cell-free DNA concentration in the sample.Reviewer’s comment: The methods also refer to the removal of possible “false-positive calls” from common environmental contaminants – can any examples of organisms that would fit these criteria be provided? Since immunocompromised patients may be at risk of infection from uncommon organisms associated with their environment (particularly uncommon moulds), it is important to understand what may not be reported. Author’s response: The manuscript was revised to provide further clarity on process to guarantee that the Karius Test does not include frequently observed environmental contaminants. Here, it is important to mention that those taxa are not removed on a case-by-case basis but are completely out of scope for the whole test.Reviewer’s comment: It’s not completely clear from the text whether results of NGS testing were used for clinical care. Although in most cases it is stated that an established diagnosis of infection was made prior to NGS testing, at least two patients had negative conventional testing and were reported to respond to therapy directed at the organisms identified by NGS. Were these patients responding to empiric treatment, or did NGS results direct the treatment? Author’s response: Two aspects of the study should be considered here. i) This was a pilot, proof-of-concept, study in which the majority of patients had an established diagnosis prior to Karius test. ii) We found that NGS correlated well with results from standard diagnostic evaluation. Consequently, the NGS results did not largely influence clinical decision making in this cohort except perhaps in patient #5 where adenovirus PCR in blood was ordered (to confirm DNAemia and monitor viral kinetics which often guide initiation of antiviral therapy) after NGS yielded adenovirus; and in patient #8 in whom clinical presentation was very suggestive of gastrointestinal sepsis due to bacterial translocation in the setting of GVHD, and although blood cultures were negative decision was made to continue empiric antibiotic therapy to complete a course in view of clinical improvement and results of NGS testing. Reviewer’s comment: Several places in the manuscript refer to the abdominal infection in the kidney transplant patient as “deep-seated,” but only once is the more medically precise term “abscess” used to describe this infection. Also, can more details about this infection be provided? This is important as the patient appears to have had negative testing (unless the detection limit was lowered below that which the assay typically uses), providing additional clues to situations in which NGS testing may provide false negative results (more on this in the next comment). Author’s response: We edited the abstract and manuscript to avoid the term “deep-seated”. This was a 65-year-old patient who presented with a 1-month history of fever, generalized body aches, malaise and abdominal pain. Ten months prior, the patient received a kidney transplant complicated with perinephric abscess due to Aspergillus fumigatus that required multiple abdominal washouts and several months of antifungal therapy. Namely, at the time of admission when NGS was sent, the patient had received >6 months of voriconazole (serum levels: 0.5-6.1 mcg/ml; target: 1.5-5 mcg/ml), and micafungin was added for persistent fungal infection. We have added some of this clinical information to the footnote of Table 1. At the time of blood sample for NGS the patient was receiving dual antifungal therapy. We suspect this is the reason why the Karius test was negative. We describe the possibility of false negative results in patients receiving antifungal therapy in the discussion: “prolonged antifungal therapy prior to sample collection (e.g., >7-14 days) might interfere with detection of fungal DNA.”Reviewer’s comment: All of the patients had received some antibiotic (antibacterial and/or antifungal) treatment at the time of NGS testing, and one of the more impressive and useful aspects of the test is that it may be able to detect organisms in the setting of effective treatment. However, although a duration of treatment is reported in Table 1, whether this treatment would have been effective against the organism identified is not clear, complicating the interpretation of the data. For example, the Discussion notes that “several months” of treatment had been given to the patient with the Aspergillus abscess prior to NGS testing, but it is not totally clear if it was all directed against Aspergillus – if so, the negative result is less concerning (and the ability to detect the organism below the reportable threshold is still impressive). Similarly, did patient 10 receive 129 days of antifungal treatment with anti-Aspergillus activity prior to his positive test? Although it is understandable why prophylactic treatment would not generally be reported for all patients, was patient 4 receiving any prophylaxis against Pneumocystis? Author’s response: Your point is well taken. The kidney transplant patient received >6 months of anti-Aspergillus therapy and was receiving combination of voriconazole plus micafungin at the time of sample collection for NGS testing. Patient 10 had indeed received 129 days of anti-mold prophylaxis/empiric treatment with various agents (including posaconazole, isavuconazole, liposomal amphotericin B [L-AmB]), and was receiving combination of isavuconazole, L-AmB and micafungin at the time of NGS testing. We believe significant fungal burden in the setting of refractory leukemia and prolonged neutropenia facilitated the detection of fungal DNA despite triple antifungal therapy. Patient 4 was not receiving prophylaxis against Pneumocystis (atovaquone had been discontinued 3 months prior presentation since CD4>400) but had received 3 days of TMP-SMX treatment dose at the time of NGS testing. We have edited the discussion to clarify that all patients (except those with viral infections) were on active antimicrobial therapy at the time of NGS testing.Reviewer’s comment:There are a handful of fairly minor editorial corrections also, including: On page 4 the “clinically reportable range” is referred to as “our” clinically reportable range – although it is acknowledged that three of the authors are from the company which performed the NGS testing, given that certain details of the testing are ultimately proprietary perhaps a more generic statement (as simple as “the clinically reportable range”) would be preferred. Author’s response: This has been modified. Reviewer’s comment: In that same paragraph, although the Mandell textbook is considered by many to be the “go-to” reference for clinical infectious diseases, perhaps it could be referred to as “a clinical infectious diseases reference textbook” or some less subjective phrase? Also, it appears the 8th edition is being referenced, which I believe is actually from 2015 (not 2014)? Author’s response: Text has been modified. 2014 is correct.Reviewer’s comment: The third paragraph in the left column on page 7 contains the phrase “there is limited data…” – as data is plural this should be corrected to “there are limited data…”. Author’s response: Thank you. This grammar mistake has been corrected in the revised version." } ] } ]	1	https://f1000research.com/articles/8-1194
https://f1000research.com/articles/9-4/v1	06 Jan 20	{ "type": "Research Article", "title": "A phase 2 open-label study of carboplatin in combination with gemcitabine as a dose-dense schedule in patients with locally advanced or metastatic breast cancer that are resistant to anthracyclines and taxanes", "authors": [ "Christopher Mansbridge", "Peter Simmonds", "Nicholas Murray", "Andrew Davies", "Louise Stanton", "Fay Chinnery", "Caroline Archer", "Peter Barrett-Lee", "Tamas Hickish", "Simon Crabb", "Christopher Mansbridge", "Peter Simmonds", "Nicholas Murray", "Andrew Davies", "Louise Stanton", "Caroline Archer", "Peter Barrett-Lee", "Tamas Hickish", "Simon Crabb" ], "abstract": "Background: Anthracycline- and taxane-based regimens form the mainstay of chemotherapy treatment in metastatic breast cancer. In patients who develop resistance to these agents, management options are limited and there is no standard of care. Thus, investigation into other chemotherapeutic agents is warranted. Methods: In this non-randomised prospective trial, patients with human epidermal growth factor 2 (HER-2)-negative locally advanced or metastatic breast cancer that were anthracycline- and taxane-resistant were treated with carboplatin at a dose equivalent to an area under the concentration–time curve of 4.5 mg/ml.min on day 1 and gemcitabine 1500 mg/m2 on day 2 of every 2-week cycle. The primary end point was overall response rate. Results: A total of five patients were enrolled prior to early termination due to difficulty in recruitment. The principal reason for recruitment difficulty was mandating anthracycline and taxane pre-treatment and HER-2 negativity. One patient had a complete response, one had a partial response, one had stable disease and two had progressive disease. Grade 4 neutropenia occurred in two patients. Conclusions: In this patient population, inclusion criteria that are too stringent may result in difficulties reaching recruitment targets. Carboplatin in combination with gemcitabine appears to be a safe option for treatment of patients with locally advanced or metastatic breast cancer. Due to the small sample size, it is not possible to draw firm conclusions regarding efficacy from this trial. Registration: EU Clinical Trials Register ID 2005-005164-83, registered on 10 April 2006.", "keywords": [ "Metastatic breast cancer", "carboplatin", "gemcitabine", "chemotherapy" ], "content": "Introduction\n\nBreast cancer is the most prevalent cancer in women, with over 1.5 million cases diagnosed annually worldwide1. Of these patients, 20 to 50% will develop metastatic disease2, with which only 25% will survive to 5 years3. Anthracycline- or taxane-based regimens form the mainstay of chemotherapy treatment in metastatic breast cancer; however, after failure of these first-line therapies, management of progressive disease is difficult and response rates for salvage therapies are as low as 25%4–6. Hence, further investigation into alternative chemotherapy regimens is required.\n\nGemcitabine is a pyrimidine analogue and antimetabolite drug with proven anti-tumour activity and tolerability in metastatic breast cancer7. In addition, gemcitabine is an excellent agent in polychemotherapy, as demonstrated by its potential for synergistic interactions with platinum agents8,9. Gemcitabine has been used in combination with platinum agents extensively in non-small cell lung cancer (NSCLC) with good results10 and, in this combination, carboplatin appears to be better tolerated than cisplatin, with similar efficacy rates11.\n\nWe proposed a prospective phase II trial to assess the efficacy of gemcitabine in combination with carboplatin in patients with human epidermal growth factor 2 (HER-2) negative, locally advanced or metastatic breast cancer that were anthracycline- and taxane-resistant.\n\n\nMethods\n\nPatients with locally advanced or metastatic breast cancer that had previously been treated with anthracyclines and taxanes were eligible to be screened for entry into the study. The protocol was approved by an independent research ethics committee and all patients gave written informed consent prior to enrolment. Research was conducted in accordance with the Good Clinical Practice guidelines. Consent materials and the full protocol are available as Extended data12.\n\nPatients were approached opportunistically within the hospital setting and screened for eligibility by hospital research staff to ensure all inclusion and exclusion criteria were met. Clinicians sought informed consent to enter the trial from a patient only after the patient had received a full explanation of the trial, had read the PIS and had enough time to consider taking part. Data were collected at the investigational site.\n\nPatients were eligible if they had histologically proven HER-2-negative breast cancer and a diagnosis of locally advanced or metastatic breast cancer, and had previously received treatment with an anthracycline and a taxane either as neoadjuvant, adjuvant or metastatic therapy. Patients were not eligible if they had received more than one prior course of chemotherapy for metastatic disease. Prior hormonal or immunotherapy was allowed, provided anti-tumoral hormonal therapy was terminated prior to enrolment. Prior radiotherapy was also allowed, providing <25% of the bone marrow was treated, the radiotherapy was completed >4 weeks prior to enrolment, the whole pelvis was not irradiated and the patient had recovered from the acute toxic effects. Irradiated lesions were not included as sites of measurable disease.\n\nPatients were required to have at least one measurable site of disease, defined as a lesion that could be accurately measured in at least one dimension as 2 cm or greater with conventional techniques or as 1 cm or greater with spiral CT scan. Palpable disease was acceptable. Where there was a solitary site of recurrence, histological or cytological confirmation was required.\n\nOther eligibility criteria included: age ≥18 years; Eastern Cooperative Oncology Group (ECOG) performance score ≤1; minimal life expectancy of 12 weeks; adequate bone marrow reserve; adequate renal and hepatic function.\n\nPatients were not eligible for enrolment if they: were pregnant, breast-feeding or refused approved contraception if of reproductive potential; had serious medical or psychiatric illness or serious active infection; had a history of a second primary malignancy other than cervical carcinoma in situ, nonmelanomatous carcinoma of the skin or other malignancy treated at least 5 years previously with no evidence of recurrence; had NCIC Common Toxicity Criteria (CTC) grade 2 peripheral neuropathy; or intended to start or stop bisphosphonate therapy within 4 weeks of enrolment. Prior administration of gemcitabine, cisplatin or carboplatin, or concomitant anti-cancer treatment was not permitted. Additionally, patients who received cytotoxic chemotherapy within the preceding 21 days or a drug without regulatory approval within the preceding 30 days prior to enrolment were not eligible.\n\nWithin the 2 weeks prior to commencing treatment, pre-trial investigations were performed. These included taking haematological samples for full blood count, serum biochemistry, hepatic function and estimation of glomerular filtration rate by Cockcroft Gault calculation, and performing a computed tomography (CT) scan of the chest, abdomen and pelvis.\n\nPatients were reviewed regularly throughout treatment. Prior to each 2-week cycle, haematological samples were obtained for full blood count, serum biochemistry and estimation of glomerular filtration rate by Cockcroft Gault calculation. In addition, patients were also required to undergo a physical examination and complete an ECOG performance questionnaire and toxicity assessment.\n\nTo assess tumour response, CT scanning of the chest, abdomen and pelvis was performed after cycles 3, 6 and 9 (where applicable). The longest diameters of target lesions were recorded (maximum of five lesions per organ and 10 lesions in total). Lesions were selected on basis of their size (lesions with the longest diameter) and suitability for accurate repeated measurements. Response assessment was based on the Response Evaluation Criteria in Solid Tumours (RECIST) criteria and classified as a complete response (disappearance of all target lesions), partial response (at least 30% decrease in the sum of the longest diameter of target lesions), progressive disease (at least 20% increase in the sum of the longest diameter of target lesions or the appearance of one or more new lesions) or stable disease (neither sufficient shrinkage to qualify for partial response nor sufficient increase to qualify for progressive disease). Overall response rate was defined as the proportion of patients exhibiting a complete or partial response at the end of treatment.\n\nPatients were followed up to obtain data on overall survival (time from date of study entry to date of death from any cause), time to progression (time from date of study entry to date of documented progression or death from any cause), duration of response (time from first documented tumour response to the date of disease progression) and time to treatment failure (time from date of study entry to the date of premature discontinuation of study treatment for any reason, or death from any cause, whichever was sooner).\n\nAll patients received intravenous carboplatin (dissolved in 250ml 5% dextrose) at a dose consistent with a target area under curve (AUC)13 of 4.5 mg/ml.min given over 30 minutes on day 1, and intravenous gemcitabine 1500 mg/m2 (up to a maximum dose of 2000 mg) at a fixed dose rate (FDR) of 10 mg/m2/min on day 2 of every 2-week cycle. This biweekly schedule has been shown to be possible in NSCLC14, where a FDR of gemcitabine15,16 and preceding gemcitabine dosing with carboplatin17 both seem to result in greater efficacy. Body surface area was calculated on each patient’s actual height and weight at the commencement of treatment.\n\nThe target number of treatment cycles was six. Patients received three treatment cycles initially, followed by a CT scan of the chest abdomen and pelvis. Patients without disease progression then received three further treatment cycles. Patients with an ongoing response as demonstrated by a further CT scan after cycle 6 were eligible to be offered an additional three cycles at the investigators’ discretion. Study treatment could be discontinued at any point due to: patient request; recurrent grade 3 or 4 treatment-related toxicity despite dose reduction; allergic reaction to a study drug; evidence of disease progression after two or more cycles of treatment; if the patient became pregnant; or if the responsible clinician felt it was not in the patients best interests to continue on study treatment.\n\nDoses were modified based on absolute neutrophil counts (ANC), platelet counts and non-haematological toxicity assessments prior to each cycle. Doses of both gemcitabine and carboplatin were reduced to 80% of the starting dose when ANC decreased to <1000/mm3 or after a first episode of uncomplicated febrile neutropenia. After a first episode of febrile neutropenia with hypotension or requiring hospital admission for more than 4 days, or a second episode of uncomplicated febrile neutropenia, doses were reduced to 60% of the starting dose or treatment was stopped if there was no evidence of response or the patient was not fit for further chemotherapy. Treatment was discontinued after a third episode of uncomplicated febrile neutropenia, or after a second episode of febrile neutropenia with hypotension or requiring hospital admission for more than 4 days.\n\nWhen platelet counts decreased to <100,000/mm3, cycles were delayed until platelets recovered (>100,000/mm3). After a second episode of thrombocytopenia (<100,000/mm3), doses of both gemcitabine and carboplatin were reduced to 80% of the starting dose. The dose was further reduced to 60% of the starting dose after a third episode. After a first episode of grade 4 thrombocytopenia or thrombocytopenia-related bleeding, doses of both gemcitabine and carboplatin were reduced to 80% of the starting dose; and this was further reduced to 60% of the starting dose after a second episode.\n\nClinical assessment of non-haematological toxicity was performed prior to each cycle. Doses were reduced to 80% of the starting dose for CTC grade 3 or 4 toxicities, or to 60% of the starting dose for CTC grade 4 toxicities. In patients with pneumonitis grade 2 or greater, gemcitabine was discontinued. Treatments could also be delayed by up to 2 weeks for any reason deemed appropriate for the treating clinician.\n\nThe primary endpoint was to determine the overall response rate. Secondary endpoints included overall toxicity, overall survival, time to disease progression, duration of response and time to treatment failure.\n\nA two-stage sampling design was employed18. For the purposes of this study, a response rate of less than 10% (null hypothesis) was considered to yield no advantage over existing therapies. Conversely, a response rate of 30% or greater (alternative hypothesis) was to indicate that the experimental regimen was of interest and warranted further evaluation. In order to reach a power of 0.9, the recruitment target was 35 patients.\n\nThe trial was entered into EudraCT on 10-APR-2006 (EudraCT Number: 2005-005164-83); https://www.clinicaltrialsregister.eu/ctr-search/trial/2005-005164-83/GB.\n\nThe trial received ethical approval from Southampton & South West Hampshire REC (A) (06/Q1702/46) on 15 June 2006. It received approval from the UK Medicines and Health Care Product Regulatory Agency (MHRA) to be conducted in the UK (MHRA CTA number 11709/0208/001-0002). Southampton Clinical Trials Unit (SCTU), a UK Clinical Research Collaboration-registered CTU, coordinated the trial. Southampton University Hospitals NHS Trust was the sponsor for the trial (email: R&Doffice@suht.swest.nhs.uk).\n\n\nResults\n\nIn total, five patients were recruited from two secondary care sites in the United Kingdom between 2007 and 2008. The study was terminated early due to difficulty in accruing patients in November 2008.\n\nOf the 34 patients screened, 29 (85%) did not meet the entry criteria and only two of the six participating sites were able to recruit patients. The most common reasons for screen failures included patients having had no previous adjuvant chemotherapy (31%) and HER-2 positivity (17%), as shown in Figure 1. Delays were also experienced waiting for local Research and Development departments to approve the study and not returning essential documents in a timely manner. The Trial Steering Committee reviewed the data and concluded that the trial would not complete in a reasonable timeframe. The primary endpoint was evaluated in all patients.\n\nThe median age of participants was 38 years (range 30 to 72 years), and all five patients were female. Patient characteristics at study entry are shown in Table 1.\n\nAbbreviations: ECOG PS, The Eastern Cooperative Oncology Group Performance Status score; CMF, cyclophosphamide, methotrexate and 5-fluorouracil; FEC, 5-fluorouracil, epirubicin and cyclophosphamide\n\nGemcitabine and carboplatin were administered intravenously by the Investigator or member of their clinical team to individual patients at the investigational sites. As a result, patient compliance monitoring was ensured. Patients who returned for follow up visits received study drug unless they encountered toxicity problems or their disease had progressed.\n\nThe study case report form was monitored by Southampton Clinical Trials Unit trial staff to ensure the gemcitabine and carboplatin doses were administered as scheduled.\n\nThe median number of cycles received was six (range 3 to 9 cycles). Two patients received less than 6 cycles, one due to progressive disease and one due to toxicity. One patient received 9 cycles due to a complete response.\n\nOf the received cycles, six doses (21%) of 28 doses of gemcitabine planned were reduced, and 15 doses (54%) delayed. No doses were omitted. The reason for reduction was myelosupression (thrombocytopenia and anaemia), and the main reason for delay was neutropenia. Likewise, six (21%) of the 28 doses of carboplatin planned were reduced, and 15 doses (54%) delayed. No doses were omitted. The reason for reduction was myelosupression (anaemia and neutropenia), and the main reason for delay was neutropenia.\n\nNotably, in three of five of the patients, the protocol was incorrectly followed by the investigating site and, in response to neutropenia, doses of both gemcitabine and carboplatin were delayed rather than reduced.\n\nOf the five patients who completed treatment, one patient achieved a complete response, one patient achieved a partial response, one patient had stable disease and two patients had disease progression. The patient with a complete response was lost to follow-up. All four of the remaining patients died, and the median overall survival was 6.3 months (range 5.0–31.6). The median time to treatment failure was 3.9 months (range 1.8–5.4), and the median time to progression was 4.7 months (range 1.8–31.6). For the patient with a partial response, the duration of response was 4.1 months.\n\nAll patients were evaluated for toxicity (Table 2). The predominant toxicities which occurred in all patients were neutropenia and anaemia. One patient developed neutropenic sepsis and subsequently discontinued treatment. There were no severe non-haematological toxicities.\n\nAbbreviations: CTC, NCIC Common Toxicity Criteria v3.0\n\n\nDiscussion\n\nIn this study, gemcitabine in combination with carboplatin seemed to be effective in treating a small number of HER-2-negative advanced or metastatic breast cancer patients. The study, however, closed early and did not reach the recruitment target of 35 patients to provide adequate power for statistical analysis. Hence, we are unable to draw firm conclusions regarding the efficacy of this combination from this study.\n\nThe major reason for early closure was difficulty in recruitment. It proved difficult to source patients who had had previous chemotherapy, including an anthracycline and a taxane, and who were HER-2 negative and fit to receive further chemotherapy. The entry criteria proved to be too stringent, limiting the number of eligible patients. This is important for future studies to take into consideration because, if using similar criteria, recruitment targets may not be met within a reasonable timeframe.\n\nThe response rate seen in this study was 40%. In view of the young study population who had not had a large amount of pre-treatment, a reasonable response rate was expected. There are now several other small phase II studies that have tested the combination of gemcitabine and carboplatin in pre-treated metastatic breast cancer20–24. These trials yielded lower response rates of 30–39%, with times to progression of 4.6–7.0 months. Entry criteria were generally less stringent, for example not requiring patients to be pre-treated with an anthracycline and taxane23–25 or including patients with any HER-2 status22–24, which may account for success in accruing patients to these studies.\n\nSignificant haematological toxicities were seen in this trial, with all patients developing neutropenia and anaemia, 60% and 40% of which were grade 3/4 respectively. One patient developed grade 4 febrile neutropenia. The proportion of patients developing haematological toxicities was comparable to other phase II studies testing the combination, where febrile neutropenia occurred in 10–15% of patients, grade 3/4 neutropenia in 10–58%, grade 3/4 thrombocytopenia in 9–51% and grade 3/4 anaemia in 10–27%20–24. Non-haematological toxicities were not severe.\n\nIn contrast to other phase II studies where gemcitabine has been given at 1000mg/m2 on day 1 and 8 of every 3 week cycle20–24, we gave 1500mg/m2 on day 2 of every 2 week cycle at a FDR of 10mg/m2/min, after it was suggested this may be more efficacious in NSCLC15,16. Due to the failure to meet recruitment targets and deviations from the protocol at one centre, further investigation is required to determine the efficacy of this regimen in metastatic breast cancer.\n\nIn summary, this study highlights the difficulty in accruing HER-2 negative metastatic breast cancer patients who have been pre-treated with both anthracyclines and taxanes. In addition, although firm conclusions regarding efficacy cannot be made due to insufficient power, this study suggests the combination of gemcitabine and carboplatin is able to be delivered to this patient population and may be efficacious, in line with other phase II studies which have now been conducted.\n\n\nData availability\n\nPseudonymised individual participant data (IPD) within the clinical trial dataset are available for sharing via controlled access by authorised SCTU staff (as delegated to SCTU by the trial sponsor). Data access can be requested via a SCTU Data Release application form (available from https://www.southampton.ac.uk/ctu/about/index.page); detailing the specific requirements and the proposed research, statistical analysis, publication plan and evidence of research group qualifications. Please email the completed form to the SCTU Data Release Committee Coordinator at ctu@soton.ac.uk.\n\nData access requests are reviewed against specific eligibility criteria by the SCTU data custodian and key members of the trial team, including a statistician and chief investigator or by an external Independent Review Panel. Decisions about requests are made promptly and usually no more than three months after receipt of request. Responses to all data requests, with a clear rationale for any refusals, will be sent promptly to the data requester.\n\nFigshare: A phase 2 open-label study of carboplatin in combination with gemcitabine as a dose-dense schedule in patients with locally advanced or metastatic breast cancer that are resistant to anthracyclines and taxanes. https://doi.org/10.6084/m9.figshare.11417550.v112.\n\nThe following extended data are available:\n\nGemCarbo Protocol Version 3 08-11-06.pdf\n\nPatient Informed Consent Form Version 3 08-11-06.pdf\n\nFigshare: TREND Statement for ‘A phase 2 open-label study of carboplatin in combination with gemcitabine as a dose-dense schedule in patients with locally advanced or metastatic breast cancer that are resistant to anthracyclines and taxanes’. https://doi.org/10.6084/m9.figshare.11417550.v112.\n\nExtended data and reporting guidelines are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nFerlay J, Soerjomataram I, Dikshit R, et al.: Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015; 136(5): E359–86. PubMed Abstract \| Publisher Full Text\n\nLu J, Steeg PS, Price JE, et al.: Breast cancer metastasis: challenges and opportunities. Cancer Res. 2009; 69(12): 4951–3. PubMed Abstract \| Publisher Full Text\n\nHowlader N, Noone AM, Krapcho M, et al.: SEER Cancer Statistics Review. 1975–2012. National Cancer Institute; Bethesda, MD based on November 2014 SEER data submission, posted to the SEER web site, April 2015. Reference Source\n\nPorkka K, Blomqvist C, Rissanen P, et al.: Salvage therapies in women who fail to respond to first-line treatment with fluorouracil, epirubicin, and cyclophosphamide for advanced breast cancer. J Clin Oncol. 1994; 12(8): 1639–47. PubMed Abstract \| Publisher Full Text\n\nBlum JL, Dieras V, Lo Russo PM, et al.: Multicenter, Phase II study of capecitabine in taxane-pretreated metastatic breast carcinoma patients. Cancer. 2001; 92(7): 1759–68. PubMed Abstract \| Publisher Full Text\n\nLivingston RB, Ellis GK, Gralow JR, et al.: Dose-intensive vinorelbine with concurrent granulocyte colony-stimulating factor support in paclitaxel-refractory metastatic breast cancer. J Clin Oncol. 1997; 15(4): 1395–400. PubMed Abstract \| Publisher Full Text\n\nBlackstein M, Vogel CL, Ambinder R, et al.: Gemcitabine as first-line therapy in patients with metastatic breast cancer: a phase II trial. Oncology. 2002; 62(1): 2–8. PubMed Abstract \| Publisher Full Text\n\nPerez EA: Gemcitabine and platinum combinations in patients with breast cancer previously treated with anthracyclines and/or taxanes. Clin Breast Cancer. 2004; 4(Suppl 3): S113–6. PubMed Abstract \| Publisher Full Text\n\nPeters GJ, Bergman AM, Ruiz van Haperen VW, et al.: Interaction between cisplatin and gemcitabine in vitro and in vivo. Semin Oncol. 1995; 22(4 Suppl 11): 72–9. PubMed Abstract\n\nZatloukal P, Petruzelka L: Gemcitabine/carboplatin in advanced non-small cell lung cancer. Lung Cancer. 2002; 38(Suppl 2): S33–6. PubMed Abstract \| Publisher Full Text\n\nMazzanti P, Massacesi C, Rocchi MB, et al.: Randomized, multicenter, phase II study of gemcitabine plus cisplatin versus gemcitabine plus carboplatin in patients with advanced non-small cell lung cancer. Lung Cancer. 2003; 41(1): 81–9. PubMed Abstract \| Publisher Full Text\n\nMansbridge C, Simmonds P, Murray N, et al.: A phase 2 open-label study of carboplatin in combination with gemcitabine as a dose-dense schedule in patients with locally advanced or metastatic breast cancer that are resistant to anthracyclines and taxanes. figshare. Journal contribution. 2019. http://www.doi.org/10.6084/m9.figshare.11417550.v1\n\nCalvert AH, Newell DR, Gumbrell LA, et al.: Carboplatin dosage: prospective evaluation of a simple formula based on renal function. J Clin Oncol. 1989; 7(11): 1748–56. PubMed Abstract \| Publisher Full Text\n\nMancuso A, Migliorino MR, Martelli O, et al.: A new biweekly schedule of carboplatin and gemcitabine: Phase I feasibility trial in patients with advanced non-small cell lung cancer (NSCLC). Proc Am Soc Clin Oncol. 2004; 22: 7245. Publisher Full Text\n\nTempero M, Plunkett W, Ruiz Van Haperen V, et al.: Randomized phase II comparison of dose-intense gemcitabine: thirty-minute infusion and fixed dose rate infusion in patients with pancreatic adenocarcinoma. J Clin Oncol. 2003; 21(18): 3402–8. PubMed Abstract \| Publisher Full Text\n\nCeribelli A, Gridelli C, De Marinis F, et al.: Prolonged gemcitabine infusion in advanced non-small cell lung carcinoma: a randomized phase II study of two different schedules in combination with cisplatin. Cancer. 2003; 98(2): 337–43. PubMed Abstract \| Publisher Full Text\n\nBajetta E, Stani SC, De Candis D, et al.: Preclinical and clinical evaluation of four gemcitabine plus carboplatin schedules as front-line treatment for stage IV non-small-cell lung cancer. Ann Oncol. 2003; 14(2): 242–7. PubMed Abstract \| Publisher Full Text\n\nSimon R: Optimal two-stage designs for phase II clinical trials. Control Clin Trials. 1989; 10(1): 1–10. PubMed Abstract \| Publisher Full Text\n\nEarl HM, Hiller L, Dunn JA, et al.: NEAT: National Epirubicin Adjuvant Trial--toxicity, delivered dose intensity and quality of life. Br J Cancer. 2008; 99(8): 1226–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNelli F, Moscetti L, Natoli G, et al.: Gemcitabine and carboplatin for pretreated metastatic breast cancer: the predictive value of immunohistochemically defined subtypes. Int J Clin Oncol. 2013; 18(2): 343–9. PubMed Abstract \| Publisher Full Text\n\nMaisano R, Zavettieri M, Azzarello D, et al.: Carboplatin and gemcitabine combination in metastatic triple-negative anthracycline- and taxane-pretreated breast cancer patients: a phase II study. J Chemother. 2011; 23(1): 40–3. PubMed Abstract \| Publisher Full Text\n\nChan D, Yeo WL, Tiemsim Cordero M, et al.: Phase II study of gemcitabine and carboplatin in metastatic breast cancers with prior exposure to anthracyclines and taxanes. Invest New Drugs. 2010; 28(6): 859–65. PubMed Abstract \| Publisher Full Text\n\nLaessig D, Stemmler HJ, Vehling-Kaiser U, et al.: Gemcitabine and carboplatin in intensively pretreated patients with metastatic breast cancer. Oncology. 2007; 73(5–6): 407–14. PubMed Abstract \| Publisher Full Text\n\nNasr FL, Chahine GY, Kattan JG, et al.: Gemcitabine plus carboplatin combination therapy as second-line treatment in patients with relapsed breast cancer. Clin Breast Cancer. 2004; 5(2): 117–22, discussion 123–4. PubMed Abstract \| Publisher Full Text\n\nAndres R, Mayordomo JI, Lara R, et al.: Gemcitabine/capecitabine in patients with metastatic breast cancer pretreated with anthracyclines and taxanes. Clin Breast Cancer. 2005; 6(2): 158–62. PubMed Abstract \| Publisher Full Text" }	[ { "id": "66468", "date": "27 Jul 2020", "name": "Lubna N. Chaudhary", "expertise": [ "Reviewer Expertise I am a breast medical oncologist in an academic medical center with 8+ years of clinical and translational breast cancer research including two investigator initiated clinical trials and grant fundings." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a prospective study to assess efficacy of dose dense carboplatin and gemcitabine regimen in HER2 negative metastatic breast cancer patients. Patient population is not clearly defined. Locally advanced is not clearly defined in eligibility. ER/PR status is not clearly mentioned. It is unclear from Table 1 if the patient population was triple negative since PR status is not mentioned. Also the time to metastatic disease from their adjuvant/neoadj chemo is not clear. Resistance to anthracycline/taxane is not defined. If TNBC, PD-L1 status is not mentioned.\n\nIt is unfortunate that the trial was closed due to poor accrual. The likely reason of difficulty in accrual was the mandated prior anthracycline use in metastatic population where anthracycline is not routinely used as first line therapy. Given the very small sample size, we cannot draw any statistically significant conclusions and stats analysis is not applicable. The authors have correctly given descriptive results given n=5. Please consider submitting this as a brief report instead.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "9680", "date": "23 May 2023", "name": "Fay Chinnery", "role": "Author Response", "response": "1. Patient population is not clearly defined. Response – we have provided the full inclusion / exclusion criteria to the manuscript in the protocol as an appendix to this paper. 2. Locally advanced is not clearly defined in eligibility. Response ‐ the inclusion criteria of the protocol provides more details on this “Inclusion criteria 1 states ‐“Where there is a solitary site of recurrence, histological or cytological confirmation is required.” 3. ER/PR status is not clearly mentioned. It is unclear from Table 1 if the patient population was triple negative since PR status is not mentioned. Response ‐ ER status is in table 1. Her2 positive disease (i.e. IHC 3+ or FISH/CISH positive) is an inclusion criteria. PR Status was not collected. 4. Also the time to metastatic disease from their adjuvant/neoadj chemo is not clear. Response ‐ The date of first line of treatment wasn’t collected so unable to provide more information on this. 5. Resistance to anthracycline/taxane is not defined. Response ‐ Patients will have advanced metastatic breast cancer and have been pretreated with anthracyclines and taxanes. See inclusion criteria 2 ‐ “Patients must have received prior treatment with an anthracycline and a taxane either as neoadjuvant, adjuvant or metastatic therapy. 6. If TNBC, PD‐L1 status is not mentioned. Response ‐ PD‐L1 status wasn’t collected so unable to provide. Of note this trial was undertaken before PD‐ 1/PD‐L1 directed immunotherapy had entered clinical trials for breast cancer. 7. The likely reason of difficulty in accrual was the mandated prior anthracycline use in metastatic population where anthracycline is not routinely used as first line therapy. Response – we agree with this assessment. 8. Please consider submitting this as a brief report instead. Response – we would prefer to keep the detailed report we have provided in keeping with transparency." } ] }, { "id": "135860", "date": "23 May 2022", "name": "Caroline Hamm", "expertise": [ "Reviewer Expertise I am a breast cancer medical oncologist whose research focuses triple negative breast cancer", "Innovative chemotherapy regimen development for triple negative breast cancer and clinical trials accrual." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-written prospective, multi-centre trial that unfortunately had very poor accrual. Progesterone receptor status is not mentioned and therefore difficult to define the study population. Therefore it is difficult to apply this study to current clinical practice.\n\nIt is unfortunate that only 5 patients were accrued to this study. It appears to be a well written study, open at a number of different sites. I agree with the authors that the criteria of use of a prior anthracycline and taxane in the metastatic setting likely restricted accrual. It is unusual that this study accrued fifteen years ago, and the treatment landscape was likely very different then.\nThe interest in this study would be with the toxicities of this protocol, rather than the efficacy. Was granulocyte colony stimulating factor used? This is important, since most of the delays were related to neutropenia. However, since three of the five patients, the protocol was followed incorrectly, again making it difficult to interpret toxicities and tolerability.\n\nAs the authors stated, perhaps the only learning in this protocol is that their eligibility criteria was flawed. This can guide researchers in writing future trials. In view of this, perhaps a Brief Report is more appropriate for this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "9681", "date": "23 May 2023", "name": "Fay Chinnery", "role": "Author Response", "response": "1. Was granulocyte colony stimulating factor used? Response – No we did not incorporate growth factors within this protocol. Defined criteria existed for dose reductions to mitigate for toxicities related to myelosuppression. 2. Progesterone receptor status is not mentioned and therefore difficult to define the study population. Response ‐ PR status was not collected in case report forms so not able to report. 3. Please consider submitting this as a brief report instead Response – we would prefer to keep the detailed report we have provided in keeping with transparency." } ] } ]	1	https://f1000research.com/articles/9-4
https://f1000research.com/articles/8-933/v1	21 Jun 19	{ "type": "Case Report", "title": "Case Report: Cetuximab use in advanced cutaneous squamous cell carcinoma resistant to chemotherapy", "authors": [ "Alvise Sernicola", "Salvatore Lampitelli", "Sara Grassi", "Antonio Giovanni Richetta", "Stefano Calvieri", "Alvise Sernicola", "Salvatore Lampitelli", "Sara Grassi", "Stefano Calvieri" ], "abstract": "We present the case of a 60-year-old man with unresectable cutaneous squamous cell carcinoma (cSCC) of the sternal area, which was not amenable to radiation therapy. The treatment history of this patient is remarkable as the disease had progressed through all lines of conventional therapy established in the literature. We decided to initiate treatment with epidermal growth factor receptor (EGFR) inhibitor cetuximab and we reassessed the patient after 12 weeks with a whole-body CT scan, documenting stability in the size and radiologic features of the disease. Cetuximab, like all current treatments for advanced cSCC, is administered off-label and proved effective in preventing further progression of disease in our patient.", "keywords": [ "cutaneous squamous cell carcinoma", "cetuximab", "EGFR", "non-melanoma skin cancer" ], "content": "Introduction\n\nThis case describes the effective use of cetuximab in an extensive thoracic cutaneous squamous cell carcinoma resistant to all previous lines of chemotherapy.\n\nNon-melanoma skin cancer (NMSC) is the most common malignant neoplasm affecting Caucasian individuals, the main types of which are basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). SCC has a lower incidence than BCC and the gold standard of treatment is surgical excision. Between 1–5% of SCCs exhibit biologically aggressive behavior and are resistant to surgery1.\n\nThe management of metastatic or locally advanced SCC includes radiotherapy (alone or in combination with surgery) and systemic chemotherapy, but a consensus for the treatment of cutaneous SCC (cSCC) is still lacking. cSCCs that don’t respond to conventional treatments pose a further challenge and may benefit from the use of target therapy, such as inhibitors of the epidermal growth factor receptor (EGFR) pathway and immunotherapy with checkpoint inhibitors2.\n\nCetuximab is a monoclonal antibody first approved for metastatic colon-rectal carcinoma with EGFR expression. EGFR is also overexpressed in the majority of SCCs and the drug is indicated, in association with radiotherapy, for locally advanced head-neck SCC, or with chemotherapy for recurrent/metastatic disease. It is normally used at the standard weekly dosage of 250mg/m2. Cetuximab use is currently off-label for cSCC in different skin sites. Current literature supports the use of monoclonal antibodies and oral agents targeting EGFR in advanced cSCC3.\n\n\nCase presentation\n\nA 60-year-old Caucasian man, currently unemployed, presented to our dermatology department complaining of the recurrence of a thoracic cSCC. Physical examination revealed an extensive ulcerative skin lesion of the sternal area covered by necrotic and fibrinous tissue. The patient reported intermittent pain and bleeding (Figure 1).\n\nClinical presentation before cetuximab (a) and after six (b) and 12 weeks of therapy (c).\n\nThe onset of a nodular skin lesion in the same site dated back to 2000, but an initial diagnosis of BCC was made only in 2013, when a single biopsy was performed (see Table 1 for timeline). A computerized tomography (CT) scan followed, demonstrating a high local burden of disease, with destructive osteo-muscular infiltration, preventing a surgical or radiation approach, and the patient was treated with vismodegib (150 mg daily). After 12 months of apparent clinical remission, a local relapse was observed, and the histologic examination of an excisional biopsy diagnosed SCC. Surgical removal of the tumor was not radical, and the patient was referred for adjuvant chemotherapy, failing four consecutive cytotoxic regimens, until the personal decision of the patient to withdraw from treatment. The four regimens were as follows: cisplatinum 100mg/m2 on day one with fluorouracil 1000mg/m2 on days 1–4 of 21-day cycles for three cycles; radio-chemotherapy with gemcitabine 3000mg/m2 on days one and 15 of 28-day-cycles; cisplatinum 100mg/m2 and docetaxel 75mg/m2 on day one of 21-day-cycles; and monotherapy with gemcitabine 3000mg/m2 on days one and 15 of 28-day-cycles for eight cycles.\n\nAP, anterior-posterior; BCC, basal cell carcinoma; CT, computerized tomography; SCC, squamous cell carcinoma; PD-L1, programmed cell death ligand-1.\n\nA stage III-disease (T3N0M0, Figure 2a)4 progressing through several lines of conventional chemotherapy advised the use of targeted and immunological therapies. First, immunohistochemistry for tissue levels of programmed cell death ligand 1 (PD-L1) was performed on the previous biopsy sample documenting no/low expression. We resorted to cetuximab, the use of which is off-label for cSCC. We administered cetuximab at an initial single dose of 400mg/m2, followed by 250mg/m2 every week, the standard dosing approved for SCC of the head-neck district, for seven cycles and every two weeks for six more cycles. The patient was staged after six and 12 weeks with a whole-body CT scan, documenting stability in the size and radiologic features of the disease. (Figure 2b and 2c).\n\nTherapy with cetuximab is ongoing and we plan to restage the patient after three months. Future management of our patient includes ongoing treatment with cetuximab or evaluation for therapy with programmed cell death protein-1 (PD-1) inhibitors.\n\nCT scan performed at baseline (a), after six (b) and 12 weeks of therapy (c), highlighting the anterior-posterior diameter of the tumor.\n\n\nDiscussion\n\nThe results of our report encourage the use of cetuximab in this setting. However, data on long-term efficacy is lacking and we are not able to predict duration of response.\n\nTreatment options for locally invasive or metastatic SCC include systemic chemotherapy, adjuvant chemo-radiotherapy, as well as inhibitors of the EGFR pathway and immunotherapy with checkpoint inhibitors, which are the latest additions2. Systemic drugs for the treatment of cSCC are used off-label and the established regimens, mainly cisplatinum-based combinations, are those that were administered to our patient5,6.\n\nResponse to second line-therapy, after failure of the first line regimen, is generally uncommon and evidence of prolonged survival is lacking. Prior treatment history, the patient’s general condition and toxicity profiles guide the choice of second-line cytotoxic agent. Gemcitabine has shown activity in previously treated subjects and was employed in our patient with radiotherapy and as single agent7.\n\nWe were challenged to select an effective treatment in this advanced case and resorted to EGFR inhibitor therapy on the biological notion that EGFR is overexpressed in over 90% of cSCC. A phase II study of unresectable cSCC treated with cetuximab for at least six weeks registered 25% objective response and 42% disease stabilization8. Cetuximab is approved for the treatment of locally or regional advanced SCC of the head and neck region (in combination with radiation) or for recurrent or metastatic disease (alone or in association with platinum). Its use in cSCC of other regions is currently off-label but our choice of drug was extensively supported by evidence in published literature3.\n\nA novel attractive approach is immune checkpoint inhibition in the context of cSCC, with monoclonal antibody cemiplimab currently undergoing registration for the treatment of cSCC9. Cancer immune surveillance is crucial to the development of cSCC, as demonstrated by the high frequency of cSCC in immunosuppressed patients. PD-1 inhibitors stimulate an anti-cancer immune response and their use in dermatology is established for the treatment of metastatic melanoma. In this case, PD-L1 expression was assessed through immunohistochemistry and low/no expression was found. Mucosal head-neck SCCs display high expression of PD-L1 in the majority of cases10; however, the role of tumor PD-L1 expression in predicting response to therapy has not been demonstrated and low expression does not contraindicate immunotherapy, even if PD-L1 negative tumors correlate to poorer prognosis and lower response to checkpoint inhibitors.\n\nIn accordance with published literature11, we support the necessity of taking serial biopsies of extensive epithelial neoplasms to exclude foci of multiple differentiation and believe that a focus of SCC existed prior to therapy with vismodegib and was responsible for the subsequent recurrence and evolution of disease.\n\n\nConclusions\n\nSerial biopsies are mandatory for advanced BCC candidates prior to vismodegib treatment.\n\nNo drugs are currently approved specifically for cSCC, so all treatments are administered off-label.\n\nThe potential efficacy of cetuximab is based on the biological similarity of cSCC to mucosal SCCs of the head-neck district.\n\nLow PD-L1 expression does not preclude the efficacy of checkpoint inhibitors.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "Grant information\n\nAssociazione Romana Ricerca Dermatologica covered the publication fees of this article as support to the authors.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nQue SKT, Zwald FO, Schmults CD: Cutaneous squamous cell carcinoma: Management of advanced and high-stage tumors. J Am Acad Dermatol. United States; 2018; 78(2): 249–61. PubMed Abstract \| Publisher Full Text\n\nStratigos A, Garbe C, Lebbe C, et al.: Diagnosis and treatment of invasive squamous cell carcinoma of the skin: European consensus-based interdisciplinary guideline. Eur J Cancer. England; 2015; 51(14): 1989–2007. PubMed Abstract \| Publisher Full Text\n\nJalili A, Pinc A, Pieczkowski F, et al.: Combination of an EGFR blocker and a COX-2 inhibitor for the treatment of advanced cutaneous squamous cell carcinoma. J Dtsch Dermatol Ges. Wiley/Blackwell (10.1111); 2008; 6(12): 1066–9. PubMed Abstract \| Publisher Full Text\n\nAmid M, editor: Part II Head and Neck. In: AJCC Cancer Staging Manual. 8th ed. New York, NY, USA: Springer; 2018; 53.\n\nTanvetyanon T, Padhya T, McCaffrey J, et al.: Postoperative concurrent chemotherapy and radiotherapy for high-risk cutaneous squamous cell carcinoma of the head and neck. Head Neck. United States; 2015; 37(6): 840–5. PubMed Abstract \| Publisher Full Text\n\nMartinez JC, Otley CC, Okuno SH, et al.: Chemotherapy in the management of advanced cutaneous squamous cell carcinoma in organ transplant recipients: theoretical and practical considerations. Dermatol Surg. Wiley/Blackwell (10.1111); 2004; 30(4 Pt 2): 679–86. PubMed Abstract \| Publisher Full Text\n\nRaguse JD, Gath HJ, Bier J, et al.: Gemcitabine in the treatment of advanced head and neck cancer. Clin Oncol (R Coll Radiol). England; 2005; 17(6): 425–9. PubMed Abstract \| Publisher Full Text\n\nMaubec E, Petrow P, Scheer-Senyarich I, et al.: Phase II study of cetuximab as first-line single-drug therapy in patients with unresectable squamous cell carcinoma of the skin. J Clin Oncol. United States; 2011; 29(25): 3419–26. PubMed Abstract \| Publisher Full Text\n\nPapadopoulos KP, Owonikoko TK, Johnson ML, et al.: REGN2810: A fully human anti-PD-1 monoclonal antibody, for patients with unresectable locally advanced or metastatic cutaneous squamous cell carcinoma (CSCC)—Initial safety and efficacy from expansion cohorts (ECs) of phase I study. J Clin Oncol. American Society of Clinical Oncology. 2017; 35(15_suppl): 9503. Publisher Full Text\n\nMalm IJ, Bruno TC, Fu J, et al.: Expression profile and in vitro blockade of programmed death-1 in human papillomavirus-negative head and neck squamous cell carcinoma. Head Neck. United States; 2015; 37(8): 1088–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhu GA, Sundram U, Chang ALS: Two different scenarios of squamous cell carcinoma within advanced Basal cell carcinomas: cases illustrating the importance of serial biopsy during vismodegib usage. JAMA Dermatol. United States; 2014; 150(9): 970–3. PubMed Abstract \| Publisher Full Text" }	[ { "id": "53108", "date": "03 Sep 2019", "name": "Gregory A Daniels", "expertise": [ "Reviewer Expertise Medical oncology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case of this 60 y/o man with a history of at least two non-melanoma skin cancers details the use of targeted hedgehog inhibitors for the initial BCC histology followed by cytotoxic chemotherapy and ultimately EGFR directed treatment for what now appears to be cuSCC at the same location. The authors raise the important point of re-biopsy at progression particularly in skin cancers where blended histologies or collision tumors may occur.\nThe use of Cetuximab has been a strategy for some time. The results outlined here are consistent with what is known—relatively modest clinical value. As referenced, the largest series found a relatively low response rate with modest durations for stable disease. The follow up here is short and the three month assessment is stable disease at best. Thus, the use of Cetuximab is of limited benefit as a single agent.\nAnti-PD1 therapy is referenced as an option at progression. For usual advanced cuSCC in elderly not immune suppressed patients, this is actually the preferred first line therapy. The monoclonal Cemiplimab was approved for use in Europe in July of 2019 and prior to that in the US following the NEJM publication by Migden et al July 2018.1 The field has dramatically changed and the case should be updated to reflect this change. In the absence of a contra-indication of immune therapy, anti-PD1 therapy is standard of care.\nIt would be very interesting to see an update to this case and the progress of this patient. At that point, the case would provide more information to clinicians.\nSpecific suggestions:\nInclude the NEJM paper outlining use of Cemiplimab in cuSCC not amenable to curative surgery or radiation.\n\nProvide longer follow up than the 12 weeks reported.\n\nOutline if the patient was treated with anti-PD1 therapy and why not.\n\nComment on toxicity of therapies.\n\nThe authors are correct in making the statement that PDL1 testing may not be needed for response to anti-PD1 therapy in this disease. In fact, Cemiplimab is approved without the requirement for testing.\n\nIf available, it would be interesting to see NGS on the tumor. The tumor mutation burden for cuSCC is very high with some usual mutations (i.e. p53 and NOTCH). As this was a confusion in the case, this data may help clarify the origin of this tumor.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "5075", "date": "06 Dec 2019", "name": "Alvise Sernicola", "role": "Author Response", "response": "Dear reviewer,Thank you for your very accurate review of our paper. We have carefully reviewed all of your suggestions and corrections and revised the manuscript accordingly. Our responses are given in a point-by-point manner below. We hope the revised version is now suitable for publication and look forward to hearing from you. Specific suggestions:1. Include the NEJM paper outlining use of Cemiplimab in cuSCC not amenable to curative surgery or radiation.Reference to the paper by Migden was added to the introduction: “In 2018, a study by Migden et al. dramatically changed the previous scenario establishing the new standard of care with PD-1 blockade in immune-competent patients, in the absence of contra-indications to immunotherapy (4). Anti-PD1 monoclonal antibody cemiplimab was consequently approved for use in Europe in July 2019.” 2. Provide longer follow up than the 12 weeks reported.We have provided a follow up of up to 80 weeks of treatment with cetuximab as a single agent (54 weeks) and in combination with nivolumab (26 weeks). 3. Outline if the patient was treated with anti-PD1 therapy and why not.Following progression to stage IV disease during cetuximab therapy, “Combination therapy with the addition of PD-1 blocker was planned and we employed locally available anti-PD1 monoclonal antibody nivolumab according to the following scheme: cetuximab single dose of 250mg/m2 Q2W and nivolumab single fixed dose of 240mg Q2W administered at alternating weeks.” (case presentation) 4. Comment on toxicity of therapies.Adverse events to cetuximab has been added to the case presentation: “Therapy was well tolerated, with the only complaint of an acneiform eruption, which began after one week of treatment and was managed with clindamycin 1% gel twice a day and oral minocycline 100mg twice a day for four weeks.”Toxicity has been commented in the discussion: “A diffuse papulopustular acneiform eruption is the most common cutaneous reaction pattern to EGFR inhibitors, reported in over two-thirds of treated subjects but severe in only 5-10% of cases. Cetuximab cutaneous toxicity is suggested to be a proxy for treatment response (10).” 5. The authors are correct in making the statement that PDL1 testing may not be needed for response to anti-PD1 therapy in this disease. In fact, Cemiplimab is approved without the requirement for testing.Thank you, this has been made explicit in the conclusions: “Low PDL-1 expression does not preclude the efficacy of checkpoint inhibitors; in fact, cemiplimab is approved without requirement for testing.” 6. If available, it would be interesting to see NGS on the tumor. The tumor mutation burden for cuSCC is very high with some usual mutations (i.e. p53 and NOTCH). As this was a confusion in the case, this data may help clarify the origin of this tumor.Thank you for your suggestion, NGS assessment of tumor mutation burden was not available." } ] } ]	1	https://f1000research.com/articles/8-933
https://f1000research.com/articles/7-724/v1	11 Jun 18	{ "type": "Case Report", "title": "Case Report: Large ileal intramural hematoma presenting as an intestinal obstruction in a patient on Warfarin with incidental breast cancer", "authors": [ "Maryam Alizadeh Forutan", "Fereshteh Araghian Mojarad", "Nasrin Rahmani", "Fereshteh Araghian Mojarad", "Nasrin Rahmani" ], "abstract": "Intramural hematoma of the gastrointestinal (GI) tract, which can present as abdominal pain or obstruction, can be a rare complication of oral anticoagulants, in particular Warfarin. In this case report, we describe an 81-year-old female patient presenting with abdominal pain, nausea, and vomiting with a previous history of rectorrhagia. The patient was receiving Warfarin therapy due to cardiac valve replacement for the past 8 years. Laboratory workup revealed elevated INR and anemia. Diagnosis of ileal intramural hematoma was based on ultrasound and CT scan findings. The patient was treated by conservative approaches including administration of fresh frozen plasma, cessation of oral intake, and fluid resuscitation. In CT images, a mass on the left breast and lymphadenopathy on the left axilla were also noticed. Given that most GI intramural hematomas caused by over-anticoagulation are treated non-surgically, considering a patient's drug history, especially in older patients with abdominal pain and obstruction symptoms, is of particular importance.", "keywords": [ "Gastrointestinal Tract", "hematoma", "anticoagulant therapy", "warfarin." ], "content": "Introduction\n\nAbdominal pain is a common complaint in patients referred to hospital emergency departments and varies from mild and self-limiting to severe and life-threatening conditions1,2. Depending on the location of the pain, symptoms, physical examination and medical history, a differential diagnosis is provided1. Since mortality and the rate of surgery secondary to abdominal pain is higher in elderly patients, prompt diagnosis of the causative agent is of particular importance3. Non-specific causes, gastroenteritis, irritable bowel syndrome, urologic disorders, and gastroenteritis are among the common etiologies for abdominal pain4. An uncommon reason for abdominal pain and bowel obstruction is intestinal intramural hematoma following anticoagulation therapy5,6. Warfarin, a vitamin K antagonist, is widely used to prevent thrombosis formation due to mechanical heart valves, atrial fibrillation, pulmonary embolism and deep venous thrombosis7. The occurrence of intestinal intramural hematoma secondary to anticoagulant therapy is an uncommon condition, which affects 1/2500 patients receiving Warfarin8. The small intestine is the common site affected by spontaneous intramural hematoma, and intramural hematoma of the colon and rectum are rare9. This complication is mostly treated by non-surgical approaches.\n\nThis article reports a case of a relatively large ileum intramural hematoma developed following Warfarin use and accidental detection of breast malignancy during a CT scan.\n\n\nCase presentation\n\nAn 81-year-old woman was admitted to the Mazandaran Heart Center, Sari, Iran in February, 2018 with a 4-day history of nausea, vomiting, and abdominal pain. She had been taking Warfarin (5 mg orally once a day) and aspirin (80 mg/day) for 8 years after aortic valve replacement without close monitoring and had a history of rectorrhagia caused by Warfarin toxicity one year ago. Colicky pain, an increase in bowel sounds and periumbilical tenderness without distension was determined by physical examination. The primary laboratory findings revealed anemia (Hb: 9.8 g/dl; normal value: 11.5–13.5 g/dl) and elevated INR (6; therapeutic range: 2–3.5), liver function and biochemistry tests were within normal values. Normal left ventricular systolic function, LVEF of 55–60%, septal hypertrophy, normal functioning prosthetic valve, and dilatation of the ascending aorta were reported by echocardiography.\n\nDuring an abdominal and pelvic ultrasound, long mucosal thickening possibly through the ascending and sigmoid colon was observed, suggestive of intramural hematoma. The patient was placed on nil per os, received supportive care and two units of fresh frozen plasma. Due to normal cardiac evaluation and partial relief of symptoms on the second day of admission, the patient was referred to surgical consultation with the possible diagnosis of descending colon and sigmoid intramural hematoma. For re-administration of anticoagulants, heparin infusion (1000 units/hour) began with precise monitoring of prothrombin time.\n\nOn the fourth day, CT scan was performed with intravenous and oral contrast (Figure 1) and intramural hematoma of ileum was diagnosed. During the CT, a radiologist noticed a mass on the left breast and a lymph node with a malignant feature on the left axilla. With the improvement of abdominal pain and vomiting (on the fourth day), oral feeding was resumed. After mammography and breast biopsy, pathological examination revealed invasive ductal cell carcinoma with lymphatic and perineural invasion. According to the immunohistochemistry staining, tumor cells were strongly ER positive, PR negative, 15% Ki67 positive and equivocal for HER2. The patient was referred to the oncology-hematology department to receive appropriate treatment.\n\n(A) Circumferential thickening of an ileal loop with adjacent fat stranding in Sagittal (1) and Coronal (2) view. (B) LAP on left axilla with malignant feature measuring 18×16mm (3) and 32×26 mm mass in left breast (4).\n\nThe patient was discharged 10 days after admission and Warfarin therapy (5 mg/d) was resumed. The patient was visited 2 weeks later with good general health assessment. Letrozole (2.5 mg/day) was started because the patient refused chemotherapy and radiotherapy.\n\n\nDiscussion\n\nThe most significant reason for the intestinal intramural hematoma is trauma, while non-traumatic or spontaneous causes include anticoagulation therapy, malignancies, and blood disorders, which are considered to be rare10,11. The intestinal sites involved in the intramural hematoma in order of frequency include jejunum, duodenum, and ileum12. The intramural hematoma can affect the esophagus, gastric and colon, but these cases are less prevalent. This complication is mostly associated with Warfarin and is more common in male subjects10,13,14; a patient's chief complaints are abdominal pain, nausea, vomiting, and absence of bowel movements or flatulence6,15. Hyperechoic bowel wall thickening or free fluid may be noted in ultrasound findings, nevertheless, as a non-unspecific test, normal ultrasound results cannot rule out the possible diagnosis of gastrointestinal intramural hematoma. The diagnostic key test is a CT scan, but there is no agreement in the literature concerning the use of contrast materials, as it may obscure intramural hyper-density and hemorrhage, and also increase the risk of exposure16,17. In the review of fourteen patients by Yoldaş et al, nine patients were treated non-surgically, with eight of them were on Warfarin therapy for heart disorders. Increased INR was also observed in anticoagulant receiving patients10. In addition to impaired coagulation tests, increased WBC count and anemia may also present15,18. For early diagnosis of this complication, taking a detailed medical history of anticoagulation use, especially in older patients with abdominal pain, is required. Treatment usually is conservative and includes cessation of oral anticoagulation, serum therapy, and correction of coagulation indices by vitamin K and blood products8,19,20. Accurate recognition of this complication leads to prevention of unnecessary surgical procedures and the risk of bleeding progression. There are also evidence that Warfarin may act as an anticancer agent; Warfarin was shown to lower the incidence of organ specific cancers including lung, prostate, and breast21,22. Therefore, the development of invasive breast cancer in the present case, despite the long-term use of Warfarin, is interesting.\n\nFinally, the question arises of which anticoagulant can be used as an alternative to Warfarin in patients with frequent life threatening hemorrhagic events, which requires no regular monitoring but has an acceptable efficacy.\n\n\nConsent\n\nWritten informed consent was obtained from patient during admission and follow up for the publication of the patient’s clinical information and accompanying images.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nCartwright SL, Knudson MP: Evaluation of acute abdominal pain in adults. Am Fam Physician. 2008; 77(7): 971–8. PubMed Abstract\n\nMacaluso CR, McNamara RM: Evaluation and management of acute abdominal pain in the emergency department. Int J Gen Med. 2012; 5: 789–97. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLewis LM, Banet GA, Blanda M, et al.: Etiology and clinical course of abdominal pain in senior patients: a prospective, multicenter study. J Gerontol A Biol Sci Med Sci. 2005; 60(8): 1071–6. PubMed Abstract \| Publisher Full Text\n\nViniol A, Keunecke C, Biroga T, et al.: Studies of the symptom abdominal pain--a systematic review and meta-analysis. Fam Pract. 2014; 31(5): 517–29. PubMed Abstract \| Publisher Full Text\n\nAltikaya N, Parlakgümüş A, Demir Ş, et al.: Small bowel obstruction caused by intramural hematoma secondary to warfarin therapy: a report of two cases. Turk J Gastroenterol. 2011; 22(2): 199–202. PubMed Abstract \| Publisher Full Text\n\nAvent ML, Canaday BR, Sawyer WT: Warfarin-induced intramural hematoma of the small intestine. Clin Pharm. 1992; 11(7): 632–5. PubMed Abstract\n\nSohrabi MK, Tajik A: Effective feature selection of clinical and genetic to predict warfarin dose using artificial neural network. Tehran Univ Med J. 2016; 73(12): 900–5. Reference Source\n\nLimmer AM, Clement Z: Extensive small bowel intramural haematoma secondary to warfarin. J Surg Case Rep. 2017; 2017(3): rjx044. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLobo L, Koudki R, Prasad Hl K, et al.: Colon Obstruction due to an Anticoagulant Induced Intramural Haematoma; A Rare Case Report. J Clin Diagn Res. 2013; 7(4): 739–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoldaş T, Erol V, Çalışkan C, et al.: Spontaneous intestinal intramural hematoma: what to do and not to do. Ulus Cerrahi Derg. 2013; 29(2): 72–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBirns MT, Katon RM, Keller F: Intramural hematoma of the small intestine presenting with major upper gastrointestinal hemorrhage. Case report and review of the literature. Gastroenterology. 1979; 77(5): 1094–100. PubMed Abstract\n\nBirla RP, Mahawar KK, Saw EY, et al.: Spontaneous intramural jejunal haematoma: a case report. Cases J. 2008; 1(1): 389. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDhawan V, Mohamed A, Fedorak RN: Gastric intramural hematoma: a case report and literature review. Can J Gastroenterol. 2009; 23(1): 19–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHong M, Warum D, Karamanian A: Spontaneous intramural esophageal hematoma (IEH) secondary to anticoagulation and/or thrombolysis therapy in the setting of a pulmonary embolism: a case report. J Radiol Case Rep. 2013; 7(2): 1–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBekheit M, AlaaSallam M, Khafagy P-A, et al.: Non-traumatic intramural hematomas in patients on anticoagulant therapy: Report of three cases and overview of the literature. Afr J Emerg Med. 2014; 4(4): e1–e4. Publisher Full Text\n\nAzcón FM, Martínez AM, Chinchilla AS, et al.: Findings in spontaneous intramural intestinal hematoma imaging. Rev Argent Radiol. 2016; 80(1): 39–44. Reference Source\n\nSorbello MP, Utiyama EM, Parreira JG, et al.: Spontaneous intramural small bowel hematoma induced by anticoagulant therapy: review and case report. Clinics (Sao Paulo). 2007; 62(6): 785–90. PubMed Abstract \| Publisher Full Text\n\nAbbas MA, Collins JM, Olden KW, et al.: Spontaneous intramural small-bowel hematoma: Clinical presentation and long-term outcome. Arch Surg. 2002; 137(3): 306–10. PubMed Abstract \| Publisher Full Text\n\nKones O, Dural AC, Gonenc M, et al.: Intramural hematomas of the gastrointestinal system: a 5-year single center experience. J Korean Surg Soc. 2013; 85(2): 58–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbbas MA, Collins JM, Olden KW: Spontaneous intramural small-bowel hematoma: imaging findings and outcome. AJR Am J Roentgenol. 2002; 179(6): 1389–94. PubMed Abstract \| Publisher Full Text\n\nSchulman S, Lindmarker P: Incidence of Cancer after Prophylaxis with Warfarin against Recurrent Venous Thromboembolism. Duration of Anticoagulation Trial. N Engl J Med. 2000; 342(26): 1953–8. PubMed Abstract \| Publisher Full Text\n\nHaaland GS, Falk RS, Straume O, et al.: Association of warfarin use with lower overall cancer incidence among patients older than 50 years. JAMA Intern Med. 2017; 177(12): 1774–80. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "35009", "date": "12 Sep 2019", "name": "Kevin W. Olden", "expertise": [ "Reviewer Expertise Functional gastrointestinal disorders" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case is well presented, and the clinical details are well written and meticulously enumerated. The case is relevant for practice and will benefit clinicians.\n\nThe only comment I would have is to change the word \"ileum\" on p.2 (2nd paragraph) to \"ileal\" and the word \"rectorrhagia\" to \"hematochezia\" in order to conform to the more commonly used medical terminology.\n\nThis paper is an excellent case report that presents useful information for the clinician. I would recommend acceptance if the two semantic changes were made.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "4926", "date": "16 Sep 2019", "name": "Maryam Alizadeh Forutan", "role": "Author Response", "response": "Greetings and regards.Thanks for bothering to read this case report and for taking the time to review the article.I will consider the issues raised." } ] }, { "id": "55318", "date": "21 Oct 2019", "name": "Everton Cazzo", "expertise": [ "Reviewer Expertise Gastrointestinal Surgery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and well-written case report of a disease which poses a significant difficulty in relation to its diagnosis and management.\nThe main comments which I think may improve and enrich this study are the following:\nThe discussion should include a more profound comment on the therapeutic options available for the cases when the bleeding does not stop after interrupting Warfarin, mainly radiological and surgical interventions.\n\nThe discussion should also include some comments on hematomas which arise on other segments of the gastrointestinal tract, such as stomach and duodenum, since they are close to the topic reported.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "5009", "date": "06 Nov 2019", "name": "Maryam Alizadeh Forutan", "role": "Author Response", "response": "Hello and thanks for your attention. I will definitely consider and correct the things you have mentioned." }, { "c_id": "5096", "date": "30 Dec 2019", "name": "Maryam Alizadeh Forutan", "role": "Author Response", "response": "Dear reviewer,Many thanks for your attention and points.We have revised the manuscript according to your suggestions and highlighted them." } ] } ]	1	https://f1000research.com/articles/7-724
https://f1000research.com/articles/8-2147/v1	30 Dec 19	{ "type": "Research Article", "title": "Using synthetic datasets to bridge the gap between the promise and reality of basing health-related decisions on common single nucleotide polymorphisms", "authors": [ "Thomas R. Wood", "Nathan Owens", "Nathan Owens" ], "abstract": "Background: While the academic genetic literature has clearly shown that common genetic single nucleotide polymorphisms (SNPs), and even large polygenic SNP risk scores, cannot reliably be used to determine risk of disease or to personalize interventions, a significant industry of companies providing SNP-based recommendations still exists. Healthcare practitioners must therefore be able to navigate between the promise and reality of these tools, including being able to interpret the literature that is associated with a given risk or suggested intervention. One significant hurdle to this process is the fact that most population studies of common SNPs only provide average (+/- error) phenotypic or risk descriptions for a given genotype, which hides the true heterogeneity of the population and reduces the ability of an individual to determine how they themselves or their patients might truly be affected. Methods: We generated synthetic datasets generated from descriptive phenotypic data published on common SNPs associated with obesity, elevated fasting blood glucose, and methylation status. Using simple statistical theory and full graphical representation of the generated data, we developed a method by which anybody can better understand phenotypic heterogeneity in a population, as well as the degree to which common SNPs truly drive disease risk. Results: Individual risk SNPs had a <10% likelihood of effecting the associated phenotype (bodyweight, fasting glucose, or homocysteine levels). Example polygenic risk scores including the SNPs most associated with obesity and type 2 diabetes only explained 2% and 5% of the final phenotype, respectively. Conclusions: The data suggest that most disease risk is dominated by the effect of the modern environment, providing further evidence to support the pursuit of lifestyle-based interventions that are likely to be beneficial regardless of genetics.", "keywords": [ "Genetics", "single nucleotide polymorphisms", "risk", "obesity", "methylation", "blood glucose" ], "content": "Introduction\n\nDue to decreasing costs and a move towards “personalized medicine”, the use of direct-to-consumer (DTC) genetic analyses and third party interpretation services is increasing1. Though whole genome sequencing is also increasing in popularity, most DTC products involve the analysis of common single nucleotide polymorphism (SNPs). These SNPs are then reported, either by the testing company or a third party tool that analyses the data, with specific disease risks based on published population data such as that from genome-wide association studies (GWAS). While the academic genetic literature has clearly shown that using SNPs, including polygenic risk scores (PRSs), to determine disease risk or to personalize clinical interventions is not currently possible or evidence-based, the trend for companies giving genetic-based advice on athletic ability or dietary recommendations is increasing2. These risk predictions or recommendations are generally based on population average outcomes, with the heterogeneity of a given phenotype or disease risk infrequently reported. In fact, most GWAS studies tend to only report descriptive data (e.g. mean and standard error) for a given phenotype [such as body mass index (BMI) or fasting blood glucose] within a risk genotype. By only comparing or providing group averages based on genotype, the consumer is likely to overestimate the disease risk associated with a given SNP. Presenting only simplified descriptive data, either graphically or numerically, for a given genotype gives the impression that each SNP has consistent penetrance with respect to the phenotype in question, which is known to not be the case3. Therefore, the interpretation of disease risk based on SNPs by those not involved in the original studies and without access to the original data is almost impossible.\n\nMore important than the mean population effects of a given SNP or combination of SNPs that influence a common phenotype is the likelihood of a physiologically-relevant effect in a given individual. This includes the likelihood that there is no overall effect of genotype, particularly compared to common environmental factors that drive chronic disease risk in high income countries such as diet, sleep, and exercise. In order to allow for healthcare practitioners or self-interested parties to better understand the likelihood of a given phenotype being altered by a specific genotype, we developed a method by which synthetic datasets could be generated and analyzed. This is largely possible due to the fact that the effects of SNPs on measurable phenotypes are generally considered to follow a normal distribution, with the number of alleles or weighted genetic scores being linearly associated with the target phenotype. The method outlined is not intended to be a systematic approach to the literature of SNPs and their association with disease risk, but is instead intended to give healthcare providers a simple tool with which to better understand the literature and answer the questions of their patients. Using this approach, the significant heterogeneity of population data can be better understood, particularly with respect to how a given individual may or may not display phenotypic changes based on the presence of common genotypes.\n\n\nMethods\n\nTo provide illustrative examples, individual studies and meta-analyses of per allele effects for common SNPs most strongly-associated with risk of type 2 diabetes (Melatonin Receptor 1B, MTNR1B rs10830963), obesity (Fat mass and obesity-associated protein, FTO rs9939609), and altered methylation and nutrient handling resulting in elevated homocysteine levels (Methylenetetrahydrofolate Reductase, MTHFR rs1801131 and rs1801133) were identified from a commonly-used third-party SNP analysis tool (FoundMyFitness Genetic Report) output, as well as the online SNP wiki SNPedia.com4–6. Due to the significant effect of ethnicity on SNP disease penetrance, example population data that were likely to most closely match the Anglo-Scandinavian background of the first author were used in individual examples, including data from deCODE (Iceland) and the Northern Finland Birth Cohort (NFBC), which were included in large multi-population GWAS studies4,5. According to recently-published methods suggested by Pontzer et al., published hunter gatherer data for fasting glucose were used to provide an estimate of the effect of the Western environment on fasting glucose and diabetes risk compared to a published genetic risk score7.\n\nPublished per allele or per genetic risk score means were used to construct synthetic datasets for a given phenotype. All publications assumed data were normally distributed and that per allele/genetic risk score effects were linear. If data were expressed as mean with standard error (SE) or 95% confidence interval (CI), the standard deviation (SD) was calculated using the number (N) of participants in each group, where SD=SE√N and SD=√N(width of 95% CI)/3.92. When the descriptive data were not included in the publication, as was the case for genetic risk scores associated with obesity and fasting blood glucose4,5, they were estimated from published graphs by extracting images and determining the number of pixels in each column and error bar relative to the scale bars on the axes. In all cases, enough data was included in the manuscript body to confirm that at least one of the estimated values was correctly determined using this method (such as total number of participants, or mean values in the highest or lowest genetic risk groups). For each genotype and gene, 1,000 synthetic individuals were randomly generated to re-create a normally-distributed dataset with the same mean and SD characteristics as those in the associated publication. Numbers were generated using Python 3.7 and the NumPy (1.17.0) and Pandas (0.25.0) libraries. The necessary code is available on GitHub (https://github.com/root-causing-health/SNPGaussianDistGenerator). Visual inspection of the data (Prism version 8, GraphPad Software, San Diego, CA) confirmed that they were normally distributed.\n\nEach synthetic dataset was graphically represented using a violin plot to show the full distribution of the data. Percent chance of a null effect from a risk allele was calculated by determining the percent overlap of the normal distribution of the wild type phenotype with that of a risk genotype using statistics.NormalDist in Python 3.8 Beta. The percent likelihood of the phenotype in a risk allele group being at or below the mean value of the “wild type” was also calculated, and linear regression analysis was performed to determine the percent contribution of risk alleles to a given phenotype. Again, to provide graphical and statistical examples, similar analyses were performed using published multi-SNP PRSs for type 2 diabetes and obesity4,5. As of the time of writing, perhaps the largest and most comprehensive published PRS for obesity (Khera et al., 2.1 million common SNPs in >300,000 individuals) could not be analyzed using this method, as only the mean phenotype in each decile of genetic risk is presented, with no error metric in either the text or figures8.\n\nTo encourage attempts to perform similar analyses, a number of free online tools can be used that do not require significant technical skills. After calculating mean and SD as described above, free gaussian random number generators such as from Random.org can be used to generate synthetic datasets. Though the Box-Muller transform used by this tool is unlikely to produce a truly normal distribution9, this is also unlikely to meaningfully affect the outcome. Similar online tools can be used to determine the likelihood of being at, above, or below, a given point in a normal distribution to determine null effects of a given SNP or risk score (http://onlinestatbook.com/2/calculators/normal_dist.html). Finally, free online graphing software can be used to visually represent the datasets for visual examination of variability and overlap (e.g. Plotly), and perform linear regression analyses (e.g. GraphPad).\n\n\nResults\n\nPublished meta-analyses suggest an increase in BMI of 0.3 kg/m2 per FTO rs9939609 A allele10. From this meta-analysis, data from the NFBC at 31 years of age (n=4,435) were used as a graphical example (Figure 1A)11. Mean (SD) BMI across the three genotypes was 24.12 (3.87) kg/m2, 24.43 (3.94) kg/m2, and 24.82 (3.95) kg/m2 for TT, AT, and AA respectively. In this population the risk of being overweight (BMI >25 kg/m2) was 41%, 44%, and 48%, resulting in an absolute 7% increase in risk in the TT genotype. BMI at or below the TT genotype was 47% in those with the AT genotype, and 43% in those with the TT genotype. The likelihood of null effect (percent overlap in BMI distribution of those with AT and AA genotypes compared to TT) was 96.8% and 92.8%, respectively. Therefore, only 3.2% of AT and 7.2% of AA genotypes would be expected to display any increase in BMI due to FTO genotype relative to TT. Linear regression found a significant association between number of A copies and BMI (p=0.001, R2=0.0035), suggesting that only around 0.4% of the variability in BMI is determined by FTO genotype (Figure 1B).\n\n(A) Violin plot displaying 1,000 synthetic BMI datapoints per FTO rs9939609 genotype, based on published population mean and SD values from the NFBC cohort. Percent overlap between the AT and AA normal distributions with that of the “wild type” (TT) genotype are displayed as a measure of the likelihood of these risk genotypes having no overall effect on BMI. (B) Linear regression of 1,000 synthetic BMI datapoints per FTO rs9939609 A allele copy. There was a significant association between number of A copies and BMI (p=0.001, R2=0.0035), suggesting that only around 0.4% of the variability in BMI is determined by FTO genotype.\n\nWiller et al. established a BMI genetic score using eight validated SNPs associated with BMI, weighted to effect size (with FTO rs9939609 given the largest weighting)5. This score was applied to the European Prospective Investigation of Cancer (EPIC) Norfolk cohort, where the top 1.2% of people (risk score >12) had an average BMI of 1.46 kg/m2 greater than those in the bottom 1.4% (risk score <4). However, the majority of participants had risk scores in the middle of the range (6–10), with large variability across the whole range of scores (Figure 2A). In the highest genetic risk groups (genetic scores of 11, 12, and >12), the likelihood of null effect was at least 80% (Table 1). The likelihood of null effect in the most common genetic score (score of 8, 18.4% of participants) was 88.1%. This suggests that regardless of an individual’s genetic score, there is less than a 20% chance that they will display any increase in BMI due to their score relative to those 1.4% of individuals with the lowest genetic risk. Across the entire range of scores, linear regression found a significant association between risk score and BMI (p<0.001, R2=0.018), suggesting that only around 2% of BMI is determined by the eight SNPs most significantly associated with BMI (Figure 2B).\n\nBMI genetic risk score, as developed by Willer et al.5, and risk of being overweight or obese, using population mean and SD values from the EPIC Norfolk cohort. Genetic scores of 6–10 cover around 75% of the population. The likelihood of null effect of each score was determined as the percent overlap of its normal distribution with that of the lowest risk group (score <4). Even in the highest risk groups (11, 12, <12) percent overlap was at least 80%, with only 12–17% of those with a genetic score of 6-10 predicted to have BMI affected by their genotype.\n\n(A) Violin plot displaying 1,000 synthetic BMI datapoints per group of BMI genetic risk score, as developed by Willer et al. using population mean and SD values from the EPIC Norfolk cohort. Significant variability is seen across the entire range of genetic scores, with more than 50% of individuals being overweight regardless of genotype. (B) Linear regression of 1,000 synthetic BMI datapoints per group of BMI genetic risk score, as developed by Willer et al.. There was a significant association between risk score and BMI (p<0.001, R2=0.018), with around 2% of BMI determined by the eight SNPs most significantly associated with BMI.\n\nOf the common SNPs associated with increased blood sugar, rs10830963 (C:G) has one of the largest effect sizes, with each G copy associated with around a 1.3 mg/dl increase in fasting blood glucose12. Data from the deCODE cohort (n=6,240) were used as a graphical example (Figure 3A)12. Mean (SD) fasting blood glucose across the three genotypes was 95.2 (12.8) mg/dl, 97.0 (12.8) mg/dl, and 97.9 (12.8) mg/dl for CC, CG, and GG respectively. The likelihood of null effect was 94.4% in those with the CG genotype, and 91.6% in those with the GG genotype. Linear regression found a significant association between number of G copies and fasting blood glucose (p<0.001, R2=0.01), with around 1% of the variability in blood glucose being determined by MTNR1B rs10830963 genotype (Figure 3B).\n\n(A) Violin plot displaying 1,000 synthetic glucose datapoints per MTNR1B rs9939609 genotype, based on published population mean and SD values from the deCODE cohort. Percent overlap between the CG and GG normal distributions with that of the “wild type” (CC) genotype are displayed as a measure of the likelihood of these risk genotypes having no overall effect on fasting blood glucose. (B) Linear regression of 1,000 synthetic BMI datapoints per MTNR1B rs9939609 G allele copy. There was a significant association between number of G copies and fasting glucose (p<0.001, R2=0.011), suggesting that only around 1% of the variability in fasting blood glucose is determined by MTNR1B genotype.\n\nSimilar to the approach of Willer et al., Dupuis et al. published a genetic risk score for elevated fasting blood glucose and risk of type 2 diabetes4, including MTNR1B and 15 other loci. This score was applied to the Framingham cohort, where the top 3.1% of people (risk score >22) had an average fasting blood glucose ~6 mg/dl greater than those in the bottom 4.2% (risk score <13). Similar to the obesity risk score, significant heterogeneity in blood glucose levels was seen across the range of scores (Figure 4A). The likelihood of null effect in the most common genetic score (score of 18, 14.3% of participants) was 84.5% (Table 2). In those with the highest genetic risk score (scores 21, 22, and >22), the risk of prediabetic level blood glucose (>100mg/dl) was double that of those in the lowest risk group. However, even in these groups the likelihood of a given genetic score being associated with blood sugar outside of the distribution of those in the lowest risk group was only 25.5–27.7%, suggesting that fewer than 30% of people with the highest genetic risk of prediabetes experience that risk as a disease phenotype. Across the entire range of scores, linear regression found a significant association between risk score and fasting glucose (p<0.001, R2=0.049), suggesting that around 5% of fasting glucose is determined by the 16 SNPs most significantly associated with type 2 diabetes risk (Figure 4B). By comparison to the Framingham cohort, where mean (SD) fasting blood glucose was 92.5 (8.7) mg/dl in the lowest genetic risk group, free living hunter gathers from Tukisenta and Kitava reportedly have fasting blood glucose of around 75 (8) and 65 (14) mg/dl, respectively (Figure 4C)13,14. Based on these data, the Tukisentans would have a 98.6% likelihood of having a blood sugar below the mean of those in the Framingham cohort with the lowest genetic risk score, with a 97.5% likelihood in the Kitavans, and normal distributions that display only 19.5% and 27.3% overlap with the lowest risk Framingham group. This translates to a 0.09% and 0.05% risk of prediabetic fasting blood glucose, respectively. Therefore, even in the lowest risk genetic group in the Framingham cohort, the relative risk of prediabetic fasting blood sugar levels (19.4%) is around 200–400 times higher than in hunter gatherer populations.\n\nGlucose genetic risk score, as developed by Dupuis et al.4, and risk of having prediabetes, using population mean and SD values from the Framingham cohort. Genetic scores of 16–19 cover around 52% of the population, and have around 30% prevalence of prediabetes. The likelihood of null effect of each score was determined as the percent overlap of its normal distribution with that of the lowest risk group (score <13). In those with the highest genetic risk scores (21, 22, and >22), the risk of prediabetic blood glucose (>100mg/dl) levels was double that of the lowest risk group. However, even in these groups the likelihood of a given genetic score being associated with blood sugar outside of the distribution of those in the lowest risk group was only 25.5–27.7%, suggesting that fewer than 30% of people with the highest genetic risk of prediabetes experience that risk as a disease phenotype.\n\n(A) Violin plot displaying 1,000 synthetic glucose datapoints per group of glucose genetic risk score, as developed by Dupuis et al.4, using population mean and SD values from the Framingham cohort. Significant variability is seen across the entire range of genetic scores. Risk of prediabetes (fasting glucose >100 mg/dl) increases from 19.4% to 43.5% from the lowest to highest risk group, with the most common genetic risk profiles (scores of 16–19, ~50% of individuals) having around a 30% risk of prediabetes. (B) Linear regression of 1,000 synthetic BMI datapoints per group of glucose genetic risk score, as developed by Dupuis et al.. There was a significant association between risk score and fasting blood glucose (p<0.001, R2=0.049), with around 5% of fasting glucose variability determined by the 16 SNPs most significantly associated with glucose homeostasis. (C) Violin plot displaying 1,000 synthetic glucose datapoints per group of glucose genetic risk score using population mean and SD values from the Framingham cohort, as well as using data from two hunter gatherer cohorts, the Tukisentans and Kitavans. The Tukisentans would have a 98.6% likelihood of having a blood sugar below the mean of those in the Framingham cohort with the best genetic score, with a 97.5% likelihood in the Kitavans. They display normal distributions with only 19.5% and 27.3% overlap with the lowest risk Framingham group and 0.09% and 0.05% risk of prediabetic fasting blood glucose, respectively. Even in the lowest risk genetic group in the Framingham cohort, the estimated prevalence of prediabetic fasting blood sugar levels (19.4%) is around 200–400 times higher than in hunter gatherer populations.\n\nTwo common polymorphisms in the MTHFR gene, which alter in vitro enzyme activity and are associated with reduced capacity to produce 5-methyltetrahydrofolate, are frequently discussed in the popular and alternative health fields with regard to the methyl cycle and associated changes in detoxification, cellular repair, and detoxification pathways. In 1998, van der Put et al. described in vitro MTHFR activity of the most common combinations of alleles at rs1801131 and rs1801133, as well as homocysteine levels in the same participants6. In the most common genotypes, excluding 1298AA/677TT, which account for around 88% of the population on average, MTHFR function across five genotypes varies from 100% to 47.7% (Table 3). However, even in those with 47.7% function (1298AC/677CT) there is an 82.1% chance of null effect compared to 1298AA/677CC “wild type” with 100% function (Table 3). Across these common mutations, MTHFR function only explains around 1% of the variability in homocysteine levels (p<0.001, R2=0.01; Figure 5A). The addition of 1298AA/677TT, which has around 12% prevalence in the population and is associated with a 75.2% loss of MTHFR function, increases the explanation of variance to 7% (Figure 5B); however, the synthetic dataset included 6.9% negative values due to the large SD in this population. This suggests significant heterogeneity of homocysteine in those with the 677TT/1298AA genotype, which is not normally distributed. Indeed, though the percent chance of non-significant difference in homocysteine levels compared to 1298AA/677CC was only 35% in those with 1298AA/677TT, this includes a large proportion of the distribution in homocysteine levels that would be below that of the “wild type” due to the very large SD in the 1298AA/677TT group; 31.3% would be predicted to have homocysteine levels below the mean of 1298AA/677CC.\n\nAverage MTHFR function and homocysteine levels by rs1801131 (A1298C) and rs1801133 (C677T) SNP combination, as published by van der Put et al.14. Genotypes are listed in order of in vitro MTHFR enzyme function, with estimated population prevalence taken from Brown et al.15. The likelihood of null effect of each combination of MTHFR SNPs was determined as the percent overlap of its normal distribution with that with the “wild type” genotype (1298AA/677CC). Significant non-linearity between degree of MTHFR function and homocysteine is seen, with the most common genotypes, representing close to 88% of the population and 47.7%-83.2% enzyme function displaying 81-95% likelihood of null effect on resulting homocysteine levels.\n\n(A) Linear regression of 1,000 synthetic homocysteine datapoints per combination of common rs1801131 (A1298C) and rs1801133 (C677T) SNPs by in vitro MTHFR activity, excluding 1298AA/677TT. There was a significant association between MTHFR function and homocysteine (p<0.001, R2=0.01), suggesting that only around 1% of the variability in homocysteine is determined by MTHFR activity across these genotypes. (B) Linear regression of 1,000 synthetic homocysteine datapoints per combination of rs1801131 (A1298C) and rs1801133 (C677T) SNPs by in vitro MTHFR activity. There was a significant association between MTHFR function and homocysteine (p<0.001, R2=0.07); however, the large SD (66% of the mean) in those with 1298AA/677TT resulted in 6.9% of predicted homocysteine levels being negative. This suggests that homocysteine in those with 1298AA/677TT is highly-variable, non-normally distributed, and that the effects of MTHFR activity on homocysteine levels are non-linear.\n\n\nDiscussion\n\nThe increasing prevalence of DTC genetic analyses is resulting in more and more healthcare providers being asked to interpret SNP-based disease risk by their patients, or attempting to incorporate these analyses into personalized treatment approaches. Here we demonstrate that, by using simple statistical theory and synthetic datasets generated based on published population phenotypic data from well-characterized SNPs, the likelihood of any given genotype resulting in a meaningful difference in phenotype is relatively small. For individual common SNPs determined to have large effect sizes, such as FTO rs9939609 on BMI and MTNR1B rs10830963 on fasting glucose, even those with two alleles have a less than 10% chance of displaying a difference in phenotype due to significant population variability. Additionally, baseline disease risks suggest that the vast majority of health outcomes associated with common SNPs are dominated by the environment.\n\nThe best-characterized SNP associated with risk of overweight and obesity is FTO rs9939609, with an average per A allele increase in BMI of 0.3 kg/m210. However, an average population effect is less useful to an individual than the likelihood that they are going to be affected in the first place. For a single FTO A allele, this likelihood is around 3%, increasing to 7% in individuals with two A alleles, with 0.4% of overall BMI explained by FTO genotype. Though it may be the SNP most well associated with increases in BMI, the vast majority of individuals are unlikely to have their BMI meaningfully affected by their FTO SNPs. Importantly, even this negligible effect of FTO on BMI is largely dominated by the environment, with recent analyses suggesting that FTO rs9939609 genotype was not associated with BMI in those born before 194216. Similarly, analyses of both FTO rs9939609 SNPs and composite obesity genetic risk scores suggest that those who partake in regular movement or exercise (~1h of moderate-vigorous physical activity per day) have similar BMIs regardless of genetics17,18. In the well-characterized EPIC Norfolk cohort, the risk of being overweight was above 50% regardless of a genetic score consisting of the eight SNPs most tightly-associated with BMI, again suggesting a significant environmental component. Considering that current Centers for Disease Control and Prevention data suggests that 39.8% of the adult population in the United States is obese, the degree to which common genetic SNPs contribute to BMI may be statistically significant but borderline physiologically irrelevant compared to the impact of the environment. For instance, using a small PRS for obesity by Willer et al.5, our analysis suggested that around 2% of BMI determined by the eight SNPs most significantly associated with BMI. Khera et al. developed a 141-SNP PRS for obesity (that could not be analyzed here due to lack of reporting of group error/SD statistics), and even then this only explained 13.3% of phenotypic variability in bodyweight8.\n\nSimilar results to those seen with genetic obesity risk were found when analyzing genetic risk of elevated fasting blood glucose and type 2 diabetes. Of the SNPs associated with increased fasting blood glucose, MTNR1B SNP rs10830963 (C:G) has one of the largest effect sizes, with each G copy associated with around a 1.3 mg/dl increase in fasting blood glucose12. In our analysis, only 5.6% of individuals with a single G copy would be expected to experience an increase in fasting blood sugar relative to those with the CC genotype, increasing to 8.2% in homozygotes. Using the genetic risk score developed by Dupuis et al. is more predictive, with more than a doubling of risk of prediabetes in those with the highest genetic frisk score compared to those with the lowest genetic risk. However, linear regression analysis suggested that only around 5% of fasting blood glucose is determined by genetic risk. This is just very similar to the proportion of explained variance that Dupuis et al. state in their original manuscript4, which provides some support for the use of synthetic datasets when variance and absolute numbers are not provided in the published literature. More importantly, however, it’s the way in which this information is placed into the context of the consumer using DTC genetic analysis to assess disease risk. For instance, the variance in fasting blood glucose (~5%) attributed to the loci included in the genetic risk score is smaller than the variance in reproducibility of commonly-used hand held at home glucometers used to monitor blood glucose in individuals with diabetes. Any effect of genetic risk is also largely a reflection of a slight amplification of the risk associated with the Western environment. Compared to hunter gatherer populations7,13,14, fasting glucose is around 25–30 mg/dl higher even in the lowest genetic risk group, and the risk of prediabetes is 200–400 times higher. Indeed, in a recent analysis of the Bolivian Tsimane, prevalence of type 2 diabetes was 0%19, on top of which any increase in genetic risk would be essentially meaningless. Therefore, the presence of any prediabetes appears to simply be a reflection of disease risk in the US as a whole, where more than 80% are thought to have suboptimal metabolic health, including more than 50% with fasting glucose >100 mg/dl20. Based on multiple lines of evidence, close to 100% of the disease risk associated with elevated fasting blood glucose in the Western world can be attributed to the modern environment.\n\nThe concept of methylation capacity and its association with long-term health has recently gained a lot of interest in the alternative health community and popular press. As a result, DTC testing of common SNPs in the MTHFR and other related genes is being used to estimate an individual’s capacity to (re)generate methylfolate in order to guide disease risk or nutrient supplementation. One potential biomarker of methyl cycle function, including MTHFR activity, is homocysteine, which is associated with and increased risk of cardiovascular disease, dementia, and all-cause mortality when elevated21–23. Though there are multiple pathways for the metabolism of homocysteine, one is dependent on methylfolate, and homocysteine levels are often used as a proxy for the status of the folate cycle. Importantly, SNPs resulting in decreased in vitro MTHFR function are common. The “wild type” genotype 677CC/1298AA associated with 100% MTHFR function is only found in around 15% of the population15, which makes some degree of reduced MTHFR function a more representative “normal” state. In addition to this, the degree of MTHFR function appears to be only loosely associated with homocysteine levels. For instance, only 1% of homocysteine was accounted for by the five rs1801131 (A1298C) and rs1801133 (C677T) combinations that encompass 47.7–100% mean MTHFR activity. This suggests significant redundancy in the system that is unlikely to be able to inform any interventions based solely on genotype. Additionally, homocysteine levels are more likely to be determined by factors not associated with direct enzyme function, as those with the 1298AC/677CC genotype have higher MTHFR activity than 1298AA/677CT (83.2% versus 66.8% relative enzyme function), but also had higher mean homocysteine levels (13.6 μmol/L versus 12.8 μmol/L)6. The non-linearity of the association between MTHFR and homocysteine levels is typified by the 1298AA/677TT genotype, who have around 75% loss of enzyme function and 50% higher mean homocysteine levels but, importantly, display a high degree of variability and values that do not appear to be normally distributed. Therefore, any specific recommendations to this group must be based in phenotypic measurements, including individual homocysteine levels and nutrient status. Indeed, though MTHFR is associated with the folate cycle, ensuring adequate B6 and B12 may be at least as important with respect to homocysteine levels24. Homocysteine in 677TT carriers can also be significantly reduced with a small amount of supplemental riboflavin25. This again suggests that phenotypic measurements and ensuring adequate environmental/nutrient status has a much greater impact than does knowledge of genotype. However, it must be cautioned that, as yet, reducing homocysteine with nutritional supplements has not yet been shown to result robustly improve health outcomes, though there may be a small reduction in stroke risk26.\n\nThis study does have some limitations. The approach used relies on the use of both simulated and statistically-ideal normal distributions based on published descriptive data rather than the data itself. However, where the methods could be tested against known data, such as the degree to which the glucose risk score explains glucose variability, the results were very similar to the original analyses. Importantly, if this approach fails to accurately recreate datasets similar to those in the published literature, then it is likely that those datasets were not normally-distributed and the original analyses were therefore inappropriate. This is probably the case for homocysteine levels in individuals with the MTHFR 1298AA/677TT genotype based on the widely-cited study by van der Put et al.6. Though all the SNPs analyzed here have low penetrance, they were specifically chosen because they are well-characterized in multiple populations and commonly included in third party DTC analyses of consumer genetic data. Though we have only highlighted a few SNPs, the techniques applied here could be used by any practitioner or interested individual to better understand their disease or outcome risk based on common genetic SNPs. Importantly, the methods described here are not intended to include a systematic exploration of the true association between common genetic polymorphisms and disease risk, but instead provide a tool that any individual can use to better understand genetic-based risk in the context of the heterogeneity of the population. Our analysis and suggestions also do not preclude the potential future utility and application of genetics in disease risk stratification when used in combination with clinical risk factors2. For instance, those with the highest genetic risk for cardiovascular disease appear to be most likely to benefit from lipid-modifying therapies2. Khera et al. also examined 50 risk SNPs for coronary disease in over 50,000 individuals, and found that those at the greatest genetic risk received the greatest risk reduction as a result of a healthy lifestyle27. However, it is also worth mentioning that the same study showed that all groups benefited from the presence of healthy lifestyle factors regardless of genetic risk, again suggesting that an individual’s environment is the common factor driving the majority of baseline disease risk.\n\nEven though there is inherent error in our approach, it is clear that using population means to determine genetic risk and make recommendations based on genetics, as is very common in the DTC market, is likely to be highly-flawed due to inherent phenotypic variability. This includes variability in risk based on common factors such as socioeconomic status and ethnicity. For instance, FTO genotypes are associated with increased BMI in Caucasians, but not in those of African origin10. For the risk of both obesity and prediabetes or type 2 diabetes, particularly, the effect of the environment (diet, exercise, nutrient status) is likely to dominate the phenotype such that knowing about an individual’s SNPs associated with risk will have little benefit. A focus on genetic risk may indeed be detrimental due to the fact that i) thinking that you have a risk SNP can have an effect on physiology regardless of whether you have that SNP28, ii) the majority of people have average genetic risk for a given phenotype, iii) DTC genetics testing still includes significant variability and error29, iv) there is little to no evidence that specific interventions for a given common SNP have any effect on health outcomes, v) communicating genetic risk does not appear to alter health behaviors30, and vi) though statistically significant, the final effect of most SNPs on phenotype could often be considered physiologically irrelevant. These risks have generally been acknowledged by the scientific community performing genetic research2, but the over-interpretation of risk by third-parties relying on published population averages remains a significant worry, likely due to misinterpretation of the nature of the data.\n\n\nConclusions\n\nUsing simple statistical techniques, either with Python code or freely-available online tools, we have outlined a method by which healthcare providers and third-party genetic analysis tools can more accurately analyze genetic disease risk. Importantly, it is worth noting that the widely-characterized and cited SNPs for obesity, type 2 diabetes, and methylation status appear to have negligible overall effects on phenotype compared to the dominant effect of the environment.\n\n\nData availability\n\nCode used to generate synthetic datasets: https://github.com/root-causing-health/SNPGaussianDistGenerator\n\nArchived code and synthetic dataset as at time of publication: https://doi.org/10.5281/zenodo.358343931\n\nLicense: MIT", "appendix": "References\n\nGuerrini CJ, Wagner JK, Nelson SC, et al.: Who's on third? Regulation of third-party genetic interpretation services. Genet Med. 2019. PubMed Abstract \| Publisher Full Text\n\nTorkamani A, Wineinger NE, Topol EJ: The personal and clinical utility of polygenic risk scores. Nat Rev Genet. 2018; 19(9): 581–590. PubMed Abstract \| Publisher Full Text\n\nBush WS, Moore JH: Chapter 11: Genome-wide association studies. PLoS Comput Biol. 2012; 8(12): e1002822. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDupuis J, Langenberg C, Prokopenko I, et al.: New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk. Nat Genet. 2010; 42(2): 105–116. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWiller CJ, Speliotes EK, Loos RJ, et al.: Six new loci associated with body mass index highlight a neuronal influence on body weight regulation. Nat Genet. 2009; 41(1): 25–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan der Put NM, Gabreels F, Stevens EM, et al.: A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am J Hum Genet. 1998; 62(5): 1044–1051. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPontzer H, Wood BM, Raichlen DA: Hunter-gatherers as models in public health. Obes Rev. 2018; 19 Suppl 1: 24–35. PubMed Abstract \| Publisher Full Text\n\nKhera AV, Chaffin M, Wade KH, et al.: Polygenic Prediction of Weight and Obesity Trajectories from Birth to Adulthood. Cell. 2019; 177(3): 587–596 e589. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohnston D: Random Number Generators—Principles and Practices: A Guide for Engineers and Programmers. De \| G Press. 2018. Reference Source\n\nQi Q, Kilpelainen TO, Downer MK, et al.: FTO genetic variants, dietary intake and body mass index: insights from 177,330 individuals. Hum Mol Genet. 2014; 23(25): 6961–6972. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaakinen M, Laara E, Pouta A, et al.: Life-course analysis of a fat mass and obesity-associated (FTO) gene variant and body mass index in the Northern Finland Birth Cohort 1966 using structural equation modeling. Am J Epidemiol. 2010; 172(6): 653–665. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nProkopenko I, Langenberg C, Florez JC, et al.: Variants in MTNR1B influence fasting glucose levels. Nat Genet. 2009; 41(1): 77–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLindeberg S, Eliasson M, Lindahl B, et al.: Low serum insulin in traditional Pacific Islanders--the Kitava Study. Metabolism. 1999; 48(10): 1216–1219. PubMed Abstract \| Publisher Full Text\n\nSinnett PF, Whyte HM: Epidemiological studies in a total highland population, Tukisenta, New Guinea. Cardiovascular disease and relevant clinical, electrocardiographic, radiological and biochemical findings. J Chronic Dis. 1973; 26(5): 265–290. PubMed Abstract \| Publisher Full Text\n\nBrown NM, Pratt VM, Buller A, et al.: Detection of 677CT/1298AC \"double variant\" chromosomes: implications for interpretation of MTHFR genotyping results. Genet Med. 2005; 7(4): 278–282. PubMed Abstract \| Publisher Full Text\n\nRosenquist JN, Lehrer SF, O'Malley AJ, et al.: Cohort of birth modifies the association between FTO genotype and BMI. Proc Natl Acad Sci U S A. 2015; 112(2): 354–359. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVimaleswaran KS, Li S, Zhao JH, et al.: Physical activity attenuates the body mass index-increasing influence of genetic variation in the FTO gene. Am J Clin Nutr. 2009; 90(2): 425–428. PubMed Abstract \| Publisher Full Text\n\nLi S, Zhao JH, Luan J, et al.: Physical activity attenuates the genetic predisposition to obesity in 20,000 men and women from EPIC-Norfolk prospective population study. PLoS Med. 2010; 7(8): pii: e1000332. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaplan H, Thompson RC, Trumble BC, et al.: Coronary atherosclerosis in indigenous South American Tsimane: a cross-sectional cohort study. Lancet. 2017; 389(10080): 1730–1739. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAraújo J, Cai J, Stevens J: Prevalence of Optimal Metabolic Health in American Adults: National Health and Nutrition Examination Survey 2009-2016. Metab Syndr Relat Disord. 2019; 17(1): 46–52. PubMed Abstract \| Publisher Full Text\n\nFan R, Zhang A, Zhong F: Association between Homocysteine Levels and All-cause Mortality: A Dose-Response Meta-Analysis of Prospective Studies. Sci Rep. 2017; 7(1): 4769. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGanguly P, Alam SF: Role of homocysteine in the development of cardiovascular disease. Nutr J. 2015; 14: 6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith AD, Refsum H, Bottiglieri T, et al.: Homocysteine and Dementia: An International Consensus Statement. J Alzheimers Dis. 2018; 62(2): 561–570. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoll S, Varga EA: Homocysteine and MTHFR Mutations. Circulation. 2015; 132(1): e6–9. PubMed Abstract \| Publisher Full Text\n\nMcNulty H, Dowey le RC, Strain JJ, et al.: Riboflavin lowers homocysteine in individuals homozygous for the MTHFR 677C->T polymorphism. Circulation. 2006; 113(1): 74–80. PubMed Abstract \| Publisher Full Text\n\nMarti-Carvajal AJ, Solà I, Lathyris D, et al.: Homocysteine-lowering interventions for preventing cardiovascular events. Cochrane Database Syst Rev. 2017; 8: CD006612. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhera AV, Emdin CA, Drake I, et al.: Genetic Risk, Adherence to a Healthy Lifestyle, and Coronary Disease. N Engl J Med. 2016; 375(24): 2349–2358. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTurnwald BP, Goyer JP, Boles DZ, et al.: Learning one's genetic risk changes physiology independent of actual genetic risk. Nat Hum Behav. 2019; 3(1): 48–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTandy-Connor S, Guiltinan J, Krempely K, et al.: False-positive results released by direct-to-consumer genetic tests highlight the importance of clinical confirmation testing for appropriate patient care. Genet Med. 2018; 20(12): 1515–1521. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHollands GJ, French DP, Griffin SJ, et al.: The impact of communicating genetic risks of disease on risk-reducing health behaviour: systematic review with meta-analysis. BMJ. 2016; 352: i1102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOwens N: root-causing-health/SNPGaussianDistGenerator: F1000 Publication Verison (Version 1.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3583439" }	[ { "id": "58756", "date": "12 Feb 2020", "name": "Yesim Aydin Son", "expertise": [ "Reviewer Expertise Health Informatics", "Clinical Bioinformatics", "Genomic Medicine", "GWAS", "Molecular Diagnostics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nInterpretation of genomic variants in the clinic for the diagnosis of genetic diseases is a current challenge of bioinformatics and medical genomics. The evaluation of the performance of molecular diagnostics will be beneficial in practice. Even though the study addresses a timely problem, the authors did not fully present the current research in the area. Review of state of the art and discussions in the literature is missing.\n\nVariant interpretation for single-gene diseases and complex genetic diseases require different methodologies, thus present different challenges. In this study, obesity, a complex genetic phenotype, selected as the study case. However, the analysis only focuses on selected few SNPs as if these phenotypes present a single gene or multigenic inheritance.\n\nGWAS is the fundamental analysis technique for complex genetic phenotypes allowing genotyping of millions of variants from individual participants. As authors also concluded, descriptive statistics have limitations in the analysis of complex genetic diseases and identifying associated SNP profiles in post-GWAS research. In the last ten years, post-GWAS analysis based on data mining techniques are under investigation, and there is an expanding literature on using data mining approaches. When a wide set of variants (SNP profiles) selected as features in predictive studies, models only based on genomic variants can outperform phenotype-based predictions or hybrid models combining genetic, clinical, and environmental factors. In light of this information designing a study base on basic statistical approaches is a major limitation of the study.\n\nAn additional concern is the random generation of synthetic individual genotypes. Authors do not account for the linkage disequilibrium between SNPs, or population frequencies of individual SNPs while randomly generating the synthetic genotyping data.\n\nExpanding the discussion on why descriptive statistics fail for complex genetic diseases, such as obesity and type-II diabetes, and need to study risk profiles rather than single individual risk SNPs will be much more beneficial to the community.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "78395", "date": "22 Feb 2021", "name": "Oliver Pain", "expertise": [ "Reviewer Expertise Genetic epidemiology", "Psychiatric genetics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study illustrates and discusses several issues with the interpretability of genotype-based risk scores as they are often reported in relative terms, as opposed to absolute terms, and the model used to derive the risk scores may have been derived within populations that are not representative of the target individual. They illustrate these issues by simulating individual-level data based on reported summary statistics, and then comparing the rate of disease/or trait mean across genetic risk score categories. The authors report the proportion of the target sample in the highest genetic risk category showing a ‘null effect’, meaning the distribution of phenotype overlaps with the phenotype of individuals in the lowest genetic risk category. In a range of settings, the authors show that the absolute difference in risk across genetic risk categories is low, and therefore the approach used by many DTCs is misleading and potentially dangerous. The authors also make the important point that application of genetic findings to different populations may also lead to highly misleading results due to differences in the distribution of the phenotype and potentially large differences in the environmental contribution to the phenotype.\nI found study very interesting and I think nicely illustrates that genetic risk should be converted to absolute risk estimates before interpretation, and that models should not be applied to individuals that are not represented by the training sample.\nSpecific comments:\nDiscussion: “Additionally, baseline disease risks suggest that the vast majority of health outcomes associated with common SNPs are dominated by the environment.”\nI think this sentence should be reworded to reflect that variance explained by current genetic risk scores is substantially lower than unmeasured factors. I say this because it currently reads as though everything the genetic risk score cannot explain is due to environmental factors, but this unexplained variance is partly due to current genetic risk scores being unable to explain the full heritability of the outcome.\n\nDiscussion: “Our analysis and suggestions also do not preclude the potential future utility and application of genetics in disease risk stratification when used in combination with clinical risk factors”.\nI was relieved to read this sentence in the discussion. Whilst I appreciate that genetics is often miss-sold as being a powerful predictor, I felt this study generally did not reflect the useful contribution to risk prediction that genetic risk scores could provide as their variance explained increase, but more importantly, as they are used in combination with non-genetic risk factors to improve risk prediction. This study spent a lot of time saying why genetics alone and currently is a bad predictor, but only very briefly discussed to contribution genetic risk score could make.\n\nDiscussion, limitation 2: “ii) the majority of people have average genetic risk for a given phenotype”\nI found this limitation confusing. The authors state that because the majority of people have the average genetic risk, focusing on genetic risk estimates may be detrimental. I do not see how this is a limitation as it merely reflects the normal distribution of genetic risk scores.\n\nTypo: “with more than a doubling of risk of prediabetes in those with the highest genetic frisk score compared”. ‘frisk’ needs to be changed to ‘risk.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-2147
https://f1000research.com/articles/8-2143/v1	27 Dec 19	{ "type": "Research Article", "title": "Visual acuity demands of different language mediums in modern primary school classrooms in Malaysia", "authors": [ "Izzah Azreena Azizan", "Eg Yue Qi", "Sharanjeet Kaur", "Sumithira Narayanasamy", "Izzah Azreena Azizan", "Eg Yue Qi", "Sharanjeet Kaur" ], "abstract": "Background: Good visual acuity is important for children’s learning but the actual visual acuity (VA) demands of classrooms are not well defined. Methods: In total, 61 classrooms from eight primary schools were included in this study. Classrooms were divided into lower and upper primary which reflect different stages of learning. Three types of national schools were included in the study, which were National, National Types Chinese (C) and Tamil (T). Each type of school utilizes different language as the medium of teaching. The measurements conducted in each classroom were: dimensions, maximum distance a student is seated and vertical height of the distance and near target. Near working distance of 28cm was assumed. Distance and near visual acuity demands (VA) were then calculated. Results: The distance and near VA demands were 0.11 ± 0.26 logMAR and 0.24 ± 0.10 logMAR for lower primary, and 0.09 ± 0.20 logMAR and 0.24 ± 0.09 logMAR for upper primary classrooms respectively. Distance and near VA demands between both stages were not significantly different (p>0.05). The distance and near VA demands for National schools were 0.24 ± 0.17 logMAR and 0.31 ± 0.04 logMAR, National Type (C) were 0.16 ± 0.11 logMAR and 0.13 ± 0.03 logMAR, National Type (T) were 0.09 ± 0.10 logMAR and 0.12 ± 0.03 logMAR respectively. There were significant differences for both distance and near VA demands between types of schools, F(2, 58) 42.19, p = 0.00; F (2, 58) 208.35, p = 0.00 respectively. Conclusions: High levels of visual acuity for distance and near are required to meet the demands of modern classroom environments. Both National Types schools require higher VA demand compared to National schools. These findings suggest current vision screening protocols and cut off points for schools might require revision.", "keywords": [ "visual acuity", "demand", "classroom" ], "content": "\n\nGood vision has always been integral to proper education development in children1,2. In Malaysia, students are required to spend at least 752 hours per year in school3. Varying visual tasks such as viewing whiteboards, near work (reading and writing), copying from a whiteboard to workbook, and computer based classes present varying visual demands on the visual system of a student. Eye screenings are often conducted in primary schools to detect students with visual problems, however, these screenings mainly measure distance visual acuity only38. Students with uncorrected refractive errors are then referred to an eye care specialist for further assessment and management4,5. However, there is a paucity of information and guidelines for referrals. Eye screenings often do not adhere to any official guidelines6–9. Most current vision screening protocols and reference criteria use 0.20 logMAR as the cut-off point for distance visual acuity4,35. This could possibly lead to other visual issues being overlooked, such as hyperopia. Hyperopia causes symptoms such as difficulty in maintaining clear and comfortable vision when viewing at near distance, as well as headaches and visual fatigue following sustained near work36. As children usually have high accommodation43, they do not face difficulties when it comes to distance visual acuity. Hence, measuring distance visual acuity will not be sufficient in identifying refractive error37,38. Therefore, there is an urgent need to properly understand the visual demands in classrooms, especially for primary school students. It has been reported that reduced vision impacts students negatively in terms of social skills, struggling with school tasks demands, and economic survival31,32. Vision disorders in childhood may be carried into adulthood, thus affecting future education33. Guidelines for proper screening protocols needs to be set in place to reduce the number of students with visual problems being overlooked as early detection will reduce the chances of the students’ visual acuity impacting their educational success.\n\nA literature search found that only 3 studies evaluating the visual demands in primary school students have been conducted. Table 1 illustrates the distance and near VA demand for each country included in these studies.\n\nThe Malaysian education system (Ministry of Education — MOE) for primary schools provides 3 different types of primary schools, which are MOE National Primary Schools, Private Institutions Primary Schools and Primary Schools in Institutions under Other Government Agencies. MOE National Primary Schools are chosen for this study as it has the highest level of enrolment compared to the other 2 institutions13, with 2,693,318 students enrolling in MOE National Primary Schools in 201816. For the purpose of this study, the 3 schools chosen were the National (Sekolah Kebangsaan), National Type (Chinese) (Sekolah Kebangsaan Cina) and National Type (Tamil) (Sekolah Kebangsaan Tamil) as these 3 schools are the top 3 nationwide in terms of enrolment. These schools use different languages for teaching; National schools use Malay as their primary language, which uses Latin letters as the medium of language, while National Type (C) uses Chinese (Mandarin) and National Type (T) uses Tamil14.\n\nThere are 2 stages of learning for MOE National Primary Schools, Stage 1 (Tahap 1) are for year 1, 2 and 3 (7 to 9 year olds), and Stage 2 (Tahap 2) are for year 4, 5 and 615 (10 to 12 year olds). It is known that Stage 1 students spend fewer hours at school as compared to Stage 2 students, 752 hours and 800 hours each respectively17. While there is a difference in hours spent in school, MOE National Schools use the same textbooks for all subjects18. Although the same syllabuses are used, different languages are used for National Type schools, which are Mandarin and Tamil18. As previous studies only focused on Latin letters, this multicultural setting of using differing mediums of language for education could lead to differing VA demands.\n\nMandarin is used as the official medium of teaching for National Type (C) schools14. It is known to be a non-alphabetical language19. Chinese characters are either pictographs (single-body) or compounds of pictographs20. Therefore, these do not always have the regularities of Latin letters21. A character may consist of 1 stroke, will others involve more than 60 strokes, leading to wide spatial complexities22.\n\nTamil is used as the official medium of teaching for National Type (T) schools23. Tamil is one of the commonly spoken South Indian languages. Tamil script is syllabic, not alphabetic, thus are comprised of a variety of strokes, curves and dot patterns unlike Latin alphabets24. Current Tamil script consists of 12 vowels, 18 consonants and one special character, the āytam.\n\nThe aim of this study was to (1) determine the distance and near visual acuity demands of Malaysian national primary school classrooms, (2) compare the visual acuity demands between Stage 1 and Stage 2 and (3) compare between different types of national schools.\n\n\nMethods\n\nThis was a cross-sectional study conducted at schools located within Klang Valley from the month of January 2019 till April 2019. Three types of national schools were selected, namely the National, National Type (C) and National Type (T). A list of all the national schools located in Klang Valley was obtained from the Ministry of Education directory. Eight different schools were then selected from the list using the RANDBETWEEN function in Microsoft Excel 2010. Classes representing each year were also selected within each school using the RANDBETWEEN function in Excel from the list of all classrooms within each school. Year 6 student classrooms were excluded from this study as they usually stayed longer than other years. This is due to the preparation for the Primary School Achievement Test, which is a national examination taken by all students in Malaysia at the end of their sixth year in primary school before they leave for secondary school. All data was collected right after school ends.\n\nThe study was approved by the Malaysian Ministry of Education (Kementerian Pendidikan Malaysia) [reference: KPM.600-3/2/3-eras(2429)], the Education Department of the Federal Territory of Kuala Lumpur (Jabatan Pendidikan Wilayah Persekutuan Kuala Lumpur) [reference: JPNWP. 900-6/1/7 Jld 21(42)], and the UKM Ethics Committee (UKM PPI/111/8/JEP-2019-209). Written informed consent was obtained from each of the school principal prior to the study. As data collection was done after classes, there was no contact with the students. Hence, consent from students & parents were not required.\n\nThe distance VA demand in logMAR was calculated using the smallest vertical height of the teachers’ writing on the whiteboard (measured using a millimetre scale) in each classroom and its furthest viewing distance (furthest student table from the whiteboard). Smartboards were not included as majority of the schools did not have such teaching tools. Due to the variability of the handwriting on the whiteboards, a minimum of 10 samples was measured in each classroom and the average was used for the demand calculation. As letter optotypes usually have a stroke width that is one fifth of the letter height, the critical detail of the smallest vertical height of the target was arbitrarily taken as one fifth of the target height39. This means that the value of the vertical height of the target will be one fifth of its original height. An example is illustrated below:\n\nA 10mm letter is viewed at a distance of 5m. The critical detail on a letter is arbitrarily taken as 1/5 of the letter height, which will be 2mm in this example39 (Figure 1).\n\n\n\nTherefore, a 10mm target viewed at a distance of 5m corresponds to a threshold acuity demand of 0.14 logMAR.\n\nThe same method was adopted and used by Hug et al.10, Narayanasamy et al.11 and Negiloni et al.12. This calculation method was also used for the calculation of near visual acuity demand. For National schools, only the height of the smallest lowercase letter was measured, excluding the ascenders and descenders. For National Type (C), the height of the smallest stroke was measured as Chinese optotypes consists of variable numbers of strokes19. As for National Type (T), the smallest height for dots, curves or lines were measured as Tamil script consist of a mixture of these three attributes24.\n\nSimilar method was used for calculating the near VA demand, using the smallest vertical height of near learning materials and the near working distance. As ethical restrictions prevented direct interaction with the children in the school classroom, a fixed near working distance of 28cm was used. This was obtained from a pilot study that was conducted on 30 students, aged 7 to 11 years old. Each student was given reading material of N5.0 in size and their viewing distance was measured. The mean viewing distance was calculated and used as a standard distance for near VA demand calculation in this study. Previous studies have reported that in children with normal vision, the near acuity reserve varies between 2.5:1 and 8:125,42. Visual acuity reserve, which is the ratio between habitual best corrected visual acuity and the acuity demand of the visual task41 is important in enabling comfortable and fluent reading especially during sustained near tasks42. Hence, a child’s habitual near visual acuity needs to be needs to be at least 2.5 times greater than the minimum required demand for comfortable sustained performance. Using this guideline, the near VA demand values were then converted to actual near VA demands, which means that the value in logMAR will be even less.\n\nAll data was entered in Excel (Microsoft Office 2010) and statistical analysis was done using IBM Statistical Package for Social Sciences (SPSS) version 25. Descriptive analysis was performed for the distance and near visual acuity demand. Comparisons of visual acuity demands between Stage 1 and Stage 2 students and between types of schools were performed using Independent t-tests and One Way ANOVA respectively. A p-value of less than 0.05 was considered as statistically significant.\n\n\nResults\n\nA total of 61 classrooms from 8 schools were included in the study. All classrooms were rectangular in shape, using only whiteboards. There was great variation in terms of students’ desk arrangement in each classroom. The mean, standard deviation (SD) and range for the classroom dimensions (length x width), furthest table away from the whiteboard, smallest distant and near material size, distance and near VA demands for all the classrooms are presented in Table 2 (see underlying data44 for values for each classroom).\n\nOne-Sample T-Test was conducted to compare VA demands between Stage 1 and Stage 2 students. Comparisons of VA demands between stages of learning are presented in Table 3. There was no significant difference between Stage 1 (M = 0.11, SD = 0.26; M = 0.24, SD = 0.10) and Stage 2 (M = 0.09, SD = 0.21; M = 0.25, SD = 0.09) for both distance and near VA demands; t (59) = 0.34, p = 0.74 and t (59) = -0.33, p = 0.75.\n\nThe mean of the distance and near demands for each type of school were compared and presented in Table 4. One way ANOVA showed significant difference for both distance (F (2, 58) = 42.19, p = 0.00) and near (F (2, 58) = 208.35, p = 0.00) VA demands between schools. A Games-Howell post hoc test revealed that there was significant difference for distance VA demand between National (0.24 ± 0.17 logMAR, p = 0.00) and National Type (C) (-0.16 ± 0.11 logMAR, p = 0.00) schools, and between National and National Type (T) (-0.09 ± 0.10 logMAR, p = 0.00) schools. However, there was no significant difference in distance VA demand between National Type (C) and National Type (T) schools (p = 0.353). Similar results were reported for near VA demand. Post hoc test revealed there was a significant difference between National (0.31 ± 0.04 logMAR, p = 0.00) and National Type (C) (0.13 ± 0.03 logMAR, p = 0.00) schools, and between National and National Type (T) (0.12 ± 0.10 logMAR, p = 0.00) schools. No significant difference was reported between National Type (C) and National Type (T) schools (p = 0.411).\n\n\nDiscussion\n\nThis study may be the first of its kind to be done in Malaysia that evaluated the distance and near visual acuity demand for different types of national primary schools. According to the International Bureau of Education3, Malaysian primary school students spend around 80.00% to 81.25% of their weekly education in school that involves visually based tasks (education of core subjects).\n\nPrevious studies in the US reported an increase in distance VA demand with increasing grade level, from 0.70 to 1.18 logMAR for grades Kindergarten (5 years old) to 2 (7 years old), and 0.48 to 0.70 logMAR for grade 3 (8 years old) to 5 (10 years old)10. A similar study conducted in Australia showed that grade 5 and 6 students require 0.33 logMAR of distance VA demand11. While another study in India reported distance VA demand of 0.35 logMAR for grades 4 to 8 and 0.24 logMAR for grades 9 to 1212. However, there was no significant difference in distance VA demand between Stage 1 and Stage 2 students for this study, 0.11 ± 0.26 and 0.09 ± 0.21 logMAR respectively. This could be due to the teachers’ handwriting on the board being similar in size regardless of grade level. As whiteboards were the most widespread teaching medium in national schools, teachers possibly adopted similar writing sizes to reduce any visual discomfort while the students view the whiteboard.\n\nThe distance VA demand for this study was 0.10 ± 0.03 logMAR. This showed that the VA demand for Malaysian primary school students to be slightly higher compared to other previous studies. This could be due to the fact that this study included Chinese and Tamil schools, both using their respective mother tongue as the language medium for teaching, while other studies focused on Latin characters10–12. As previously mentioned, Chinese and Tamil characters tend to be more complex in nature as compared to Latin characters19,27. For Chinese characters, the dot is one type of stroke that makes up a single body character. This character could be a single body character or a compound character that is made up of multiple parts21. As for Indian characters, the aspect ratio is more than Latin characters26,28. This is due to Tamil characters having more curves, number of strokes, holes and sliding characters29. Currently, no complete hand written text recognition is available for Tamil due to the large character set, presence of vowel modifiers and compound character30. Hence, the smallest vertical height measured for both languages included the dots and strokes. This could possibly lead to the higher distance VA demand for such schools.\n\nNear VA demand for this study was reported to be 0.24 ± 0.01 logMAR. Previous studies in the US, Australia and India reported 0.96 ± 0.34 logMAR, 0.72 ± 0.09 logMAR, and 0.83 ± 0.14 logMAR each respectively for near VA demand. Again, this study showed higher near VA demand compared to other studies and could be due to the inclusion of National Type (C) and National Type (T) schools. The smallest vertical stroke measured was as small as 0.10cm, with a mean of 0.22cm. This resulted in a higher near VA demand compared to previous studies. For comparison between stages of education, both Stage 1 and Stage 2 students had demands of 0.24 ± 0.10 logMAR and 0.25 ± 0.09 logMAR and no significant difference (p = 0.75) were found between both stages. This could be due to similar writing sizes in students’ workbook writings and printing as all schools use the same textbooks but with different languages18.\n\nHowever, there is a significant difference when it comes to the comparison between National and National Type (C) schools and between National and National Type (T) schools. Differences in the smallest stroke size in their workbook could lead to the significant difference. As previously stated, both Chinese and Tamil characters are more complex compared to Latin character, consisting of dots, strokes and curves. Hence the smallest print size for National Type (C) schools were 0.10cm, with a mean of 0.13cm, for National Type (T) schools, it was also 0.10cm, with a mean of 0.16cm, National schools measured at 0.15cm, but with a mean of 0.26cm, which produced a lower near VA demand compared to the other 2 schools. But between National Type (C) and (T), there were no significant differences (p = 0.411) in terms of near VA demand. This could possibly be due to similar print size as stated previously.\n\nThis study is limited to only schools in urban areas, future studies should expand into suburban areas to observe the VA demand as compared to this study. Private institutions should also be included as different syllabus are taught in these schools, namely the British, American, Australian, Canadian and International Baccalaureatte34.\n\n\nConclusion\n\nThe distance and near VA demands for this study was reported to be 0.10 ± 0.03 logMAR and 0.24 ± 0.01 logMAR respectively. There was no significant difference of VA demands between stages of learning. However, National Type (C) and National Type (T) schools had significantly higher VA demands compared to National schools. However, there was no significant difference between National Type (C) and National Type (T) schools. Current vision screening protocols and reference criteria of using 0.20 logMAR as the cut-off point36,37 should be revised and updated. This is to ensure that children with possible refractive errors that may impact on their school performance are not excluded for further reference to primary eye care professionals.\n\n\nData availability\n\nHarvard Dataverse: Visual acuity demands of different language medium modern primary school classrooms in Malaysia. https://doi.org/10.7910/DVN/BDX8ZU44\n\nThis project contains the following underlying data:\n\nResearch Data – F1000 final.tab (Collected data from classrooms and visual acuity demand)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors thank all the participating schools.\n\n\nReferences\n\nBateman R, Garzia R, Rouse M, et al.: Vision, learning, and dyslexia. A joint organizational policy statement. American Optometric Association. 1997. Reference Source\n\nBasch CE: Vision and the achievement gap among urban minority youth. J Sch Health. 2011; 81(10): 599–605. PubMed Abstract \| Publisher Full Text\n\nInternational Bureau of Education, UNESCO: World Data on Education. 7th Edition. 2010. Reference Source\n\nChen AH, Bletthing W, Lim YY: Relating vision status to academic achievement among year-2 school children in Malaysia. Optometry. 2011; 82(5): 267–273. PubMed Abstract\n\nHashim SE, Tan HK, Wan-Hazabbah WH, et al.: Prevalence of refractive error in Malay primary school children in suburban area of Kota Bharu, Kelantan, Malaysia. Ann Acad Med Singap. 2008; 37(11): 940–946. PubMed Abstract\n\nNurul Farhana AB, Chen AH, Abdul Rahim MN, et al.: Pilot study: A review of personnel involved in school vision screening and the training module in Betong, Malaysia. Int Med J Malays. 2012; 11(2): 23–27. Reference Source\n\nChen AH, Teoh SC: Kajian mengenai tahap kesedaran penjagaan penglihatan terhadap kanak-kanak. Jurnal Kesihatan Masyarakat Isu Khas. 2002. Reference Source\n\nChen AH, Duratul AH: Tinjauan tentang program penyaringan penglihatan di sekolah rendah di Selangor dan Wilayah Persekutuan. Prosiding Persidangan Ulang Tahun ke-30 UKM. 2000; 469–478. Reference Source\n\nChen AH, Duratul AH: Tinjauan tentang peranan optometris dalam penjagaan penglihatan pediatrik di Malaysia. Buletin Kesihatan Masyarakat – pascasidang Kolokium Kebangsaan Kesiahatn Masyarakat ke-VII. Isu Khas. 2011; 230–234. Reference Source\n\nLangford A, Hug T: Visual demands in elementary school. J Pediatr Ophthalmol Strabismus. 2010; 47(3): 152–156. PubMed Abstract \| Publisher Full Text\n\nNarayanasamy S, Vincent SJ, Sampson GP, et al.: Visual demands in modern Australian primary school classrooms. Clin Exp Optom. 2016; 99(3): 233–240. PubMed Abstract \| Publisher Full Text\n\nNegiloni K, Ramani KK, Sudhir RR: Do school classrooms meet the visual requirements of children and recommended vision standards? PLoS One. 2017; 12(4): e0174983. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalaysia Educational Statistics: Quick Facts 2018. 2018. Reference Source\n\nSelvadurai S, Liu OP, Radzi MM, et al.: Debating education for nation building in Malaysia: National school persistence or vernacular school resistance? Malaysian Journal of Society and Space. 2017; 11(13): 14–23. Reference Source\n\nMalaysia Educational Statistics: Perangkaan Pendidikan Malaysia. Educational Planning and Research Division, Ministry of Education Malaysia. 2017. Reference Source\n\nMalaysia Educational Statistics: Planning and Research Division. Ministry of Education Malaysia. 2017. Reference Source\n\nSurat Pekeliling Ikhtisas KPM Bil 8 2016: Pelaksanaan KSSR (Semakan 2017) Secara Berperingkat-peringkat Mulai 2017. Kementerian Pendidikan Malaysia. Reference Source\n\nBahagian Sumber Dan Teknologi Pendidikan: Katalog Buku Teks Terbitan Baharu. Kementerian Pendidikan Malaysia. 2019. Reference Source\n\nZhang JY, Zhang T, Xue F, et al.: Legibility variations of Chinese characters and implications for visual acuity measurement in Chinese reading population. Invest Ophth Vis Sci. 2007; 48(5): 2383–2390. PubMed Abstract \| Publisher Full Text\n\nHao YM, Johnston AW: An evaluation of logMAR vision test charts for near vision using Chinese characters. Clin Exp Optom. 1997; 80(5): 178–186. Publisher Full Text\n\nLü X, Zhang J: Reading efficiency: a comparative study of English and Chinese orthographies. Lit Res Instr. 1999; 38(4): 301–317. Publisher Full Text\n\nWang CX, Lin N, Guo YX: Visual requirement for Chinese reading with normal vision. Brain Behav. 2019; 9(4): e01216. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchiffman HF: Language Shift in the Tamil Communities of Malaysia and Singapore. Southwest J Linguist. 1995; 14: 151–65. Reference Source\n\nVaradharajan S, Srinivasan K, Kumaresan B: Construction and validation of a Tamil logMAR chart. Ophthal Physl Opt. 2009; 29(5): 526–534. PubMed Abstract \| Publisher Full Text\n\nLouie-Kitchin JE, Oliver NJ, Bruce A, et al.: The effect of print size on reading rate for adults and children. Clin Exp Optom. 1994; 77(1): 2–7. Publisher Full Text\n\nNegiloni K, Mazumdar D, Neog A, et al.: Construction and validation of logMAR visual acuity charts in seven Indian languages. Indian J Ophthalmol. 2018; 66(5): 641–646. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFeldman LB, Siok WW: The role of component function in visual recognition of Chinese characters. J Exp Psychol Learn Mem Cogn. 1997; 23(3): 776–781. PubMed Abstract \| Publisher Full Text\n\nDhanya D, Ramakrishnan AG: Simultaneous recognition of Tamil and Roman scripts. Proc Tamil Internet. 2001; 26–28. Publisher Full Text\n\nJose TM, Wahi A: Recognition of Tamil Handwritten Characters using Daubechies Wavelet Transforms and Feed-Forward Backpropagation Network. Int J Comput Appl. 2013; 64(8): 26–29. Publisher Full Text\n\nWahi A, Sundaramurthy S, Poovizhi P: Recognition of handwritten Tamil characters using wavelet. Int J Comput Sci Eng Te. 2014; 5(4): 335–340. Reference Source\n\nKeller H: Helen Keller International_Refractive Error. Reference Source\n\nLucky KE, Od U, Tochi IF, et al.: Effects of reduced visual acuity on academic performance among secondary school students in South-South Nigeria. Int Journal Sci Res. 2014; 3: 328–34. Reference Source\n\nDavidson S, Quinn GE: The impact of pediatric vision disorders in adulthood. Pediatrics. 2011; 127(2): 334–339. PubMed Abstract \| Publisher Full Text\n\nKementerian Pendidikan Malaysia, Bahagian Pendidikan Swasta: Panduan Am Penubuhan Dan Pendaftaran Sekolah Antarabangsa. 2018. Reference Source\n\nBakar NFA, Chen AH: Discrepancy in the Accuracy of Vision Screening Program Performed by Allied Health Personnel in a Preschool. Pertanika J Sci Technol. 2017; 25: 151–158. Reference Source\n\nGrosverner TP: Primary Care Optometry: Anomalies of Refraction and Binocular Vision. St Louis: Butterworth-Heinemann. 1996. Reference Source\n\nO'Donoghue L, Rudnicka AR, McClelland JF, et al.: Visual acuity measures do not reliably detect childhood refractive error--an epidemiological study. PLoS One. 2012; 7(3): e34441. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeone JF, Mitchell P, Morgan IG, et al.: Use of visual acuity to screen for significant refractive errors in adolescents: is it reliable? Arch Ophthalmol. 2010; 128(7): 894–899. PubMed Abstract \| Publisher Full Text\n\nBailey IL: Visual acuity. In: Benjamin WJ, Borish IM eds. Borish’s Clinical Refraction. St. Louis: Butterworth-Heinemann. 2006; 217–246. Publisher Full Text\n\nLegge GE, Pelli DG, Rubin GS, et al.: Psychophysics of reading--I. Normal vision. Vision research. 1985; 25(2): 239–52. PubMed Abstract \| Publisher Full Text\n\nWhittaker SG, Lovie-Kitchin J: Visual requirements for reading. Optom Vis Sci. 1993; 70(1): 54–65. PubMed Abstract \| Publisher Full Text\n\nLueck AH, Bailey IL, Robert Greer OD, et al.: Magnification Needs to Optimize Reading Efficiency for. Vision Rehabilitation. 2000; 311. Reference Source\n\nSterner B, Gellerstedt M, Sjöström A: Accommodation and the relationship to subjective symptoms with near work for young school children. Ophthalmic Physiol Opt. 2006; 26(2): 148–55. PubMed Abstract \| Publisher Full Text\n\nAzizan IA: Visual acuity demands of different language medium modern primary school classrooms in Malaysia. Harvard Dataverse, V1. 2019. http://www.doi.org/10.7910/DVN/BDX8ZU" }	[ { "id": "58110", "date": "14 Feb 2020", "name": "Helle Kristine Falkenberg", "expertise": [ "Reviewer Expertise vision science", "optometry", "knowledge translation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMain findings:\nThe authors investigates the acuity demands of three different languages (Malay, Chinese, Tamil) for distance (handwriting) and near (printed) optotypes at two different school levels. Their results show that the acuity demand was approx. 0.1 logMAR for distance and 0.24 logMAR for near for both school levels, but they did find a significant difference between the different languages, where Chinese and Tamil had higher acuity demands for distance and near demands compared to Malay. They conclude that the acuity demand is higher than the common vision screening criteria, and that vision screening protocols should revise their cut off points.\n\nAbstract: The abstract is a fair description of the findings, and the conclusion is supported by the results.\n\n“Good visual acuity is important for children’s learning but the actual visual acuity (VA) demand” Move the abbreviation VA to first mention start of sentence.\n\nDistance and near visual acuity demands (VA) were then calculated. Remove (VA) or substitute visual acuity with VA before demand. VA is not the abbreviation for VA demands…\n\nDetailed comments\nThe central question raised in this clinical paper is not novel or original, but is of general interest internationally and specific interest to readers using Chinese and Tamil language, and within the scope of f1000.\n\nIntroduction The introduction covers relevant topics related to the study, but there are more citations that could be added in the first sentence “Good vision has always been integral to proper education development in children” (e.g. Quaid et al, 20131; Kulp et al, 20162, Basch et al 20113)) “This could possibly lead to other visual issues being overlooked, such as hyperopia.” Is supported (e.g. Falkenberg et al., 20194, Mayro, et al 20185).\n\nIt is not evident the authors have performed a proper literature search, “A literature search found … ”as they do not mention any details of such a search (search words and combintations, databases…), I suggest re-writing to we have only identified 3 studies…\n\nMethods and statistical analysis, and its interpretation appropriate?\n\nThe methods are well described and appropriate for the aim, there are some issues regarding the size of the sample sizes of the three schools –where there are 40 Malay (latin) classrooms, and only 10 of each Chinese and Tamil. Further, it would be useful to have all the raw data from each classroom, and not just the average for each classroom.\n\n“The critical detail on a letter is arbitrarily taken as 1/5…” Remove arbitrarily –it is explained in the paragraph above.\n\nThe calculations could be tidied up by explaining MAR in the text. It should be earlier, as the term logMAR is introduced in the paragraph above the figure.\n\nStatistics: When the sample sizes are so different between schools, it means that the one-way anova is not appropriate statistics.\n\nResults\n\nIt is not clear that the VA demands for distance and near in table 2 is the overall mean for all three schooltypes – and this is problematic as the authors find a significant difference between the Malay and Chinese/Tamil schools both for distance and near. This means that for Malay (latin), the VA demand for distance really is 0.24, not 0.1 –this is on line significant difference in VA. For near VA is 0.3, a difference of approx. 2 lines. This means that for Malay schools, most pupils will probably have good enough vision – even taking into account the near VA reserve. I think it would add to the manuscript if the authors calculated and showed what the habitual near VA should be for comfortable near work for the three schools. (For Chinese and Tamil it is unlikely the children have the required VAs…, and most are probably working close to their VA thresholds) this should be elaborated.\n\nThere seems to be a significant difference between distance and near VA demands, even if there are no difference between stage 1 and 2. There is no need for a separate table 3, as this is stated in the text. I suggest to remove.\n\nThe most interesting findings are the differences between type of schools, for both distance and near. However, there is a large difference in sample sizes, so authors should use more appropriate statistics for the analysis. Information that is in table 4 do not need to be repeated fully in the text.\n\nDiscussion: The point that there is a different demand for distance and near should be included in the discussion. Further that there is significantly higher demands for the twenty Chinese and Tamil classrooms, compared to the 41 Maylay classrooms for both distance and near VA.\n\n“This could be due to similar writing sizes in students’ workbook writings and printing as all schools use the same textbooks but with different languages”. Is this relevant as the near VA demand was printed material? Rephrase.\n\nConclusion: In my opinion, the authors should be careful to average the VA demands for distance and near across languages, as they clearly are different. The conclusion should reflect this, as the VA demands reported, are heavily influenced by the minority of the classrooms tested (the 20 Chinise/Tamil compared to the 41 Malay).\n\nTables:\nTables 1-3 could be adapted to fit in a column, and arranged more appropriately in the final version of the text for readability. significant differences should be marked in tables by e.g.*\n\nTable 2: the VA demands should be taken out of the table, as this average do not reflect correctly the sample as it is the 20 Chinese/Tamil schools that pull the average down. This should also be commented in the results, as this is not really accurately representing the data.\n\nTable 3: I suggest to remove as all information is stated in the text. If kept, the interesting thing is probably that there is a difference between distance and near –sig differences should be marked.\n\nTable 4: This is the most interesting results, please redo statistics and mark out significant differences.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "58109", "date": "17 Feb 2020", "name": "Rachapalle Reddi Sudhir", "expertise": [ "Reviewer Expertise Public health Ophthalmology", "Big data", "epidemiology", "artificial intelligence", "cornea and refractive surgery", "clinical trials" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is well written and relevant in view of identifying the minimum visual acuity demand in schools. Previous articles in this subject used only English language. The need for measurement of minimum visual acuity demand for different languages and its influence is well highlighted in this article.\n\nVery high demand in these schools compared to published literature is a surprise and the authors attribute it to language, any other factors need to be reviewed.\nUse of white boards and the width of the stroke on white boards are smaller compare to blackboards which would be one of the other important factor apart from the language. There is no literature available on the comparison of white board vs black board or smart boards (computer based class rooms) and it is missing in this paper also.\n\nSample size justification is not provided why did they choose randomly 8 schools only.\n\nAuthors conclude the need for revision of cut off for vision screening, they could use the data and suggest what could be the ideal cut off based on the study results. A discussion on the same and the implications of reducing the cut off like adding more burden on school screening and more students need refraction need to be highlighted.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-2143
https://f1000research.com/articles/8-1738/v1	10 Oct 19	{ "type": "Research Article", "title": "Altitude and its inverse association with abdominal obesity in an Andean country: a cross-sectional study", "authors": [ "Jaime Pajuelo-Ramírez", "Harold Torres-Aparcana", "Rosa Agüero-Zamora", "Antonio M. Quispe", "Jaime Pajuelo-Ramírez", "Rosa Agüero-Zamora", "Antonio M. Quispe" ], "abstract": "Background: Abdominal obesity represents an accurate predictor of overall morbidity and mortality, which is worrisome because it is also continuously increasing across Andean countries. However, its relationship with altitude remains unclear. The objective of this study was to assess the association between altitude and abdominal obesity in Peru, and how sociodemographic variables impact this association. Methods: We estimated the prevalence of abdominal obesity in Peru and analyzed its association with altitude using the data from the 2012-2013 National Household Survey (ENAHO). During this survey, a representative sample of Peruvians was screened for abdominal obesity, using waist circumference as a proxy, and the Adult Treatment Panel III guidelines cutoffs. Results: Data were analyzed from a sample of 20,489 Peruvians (51% male). The prevalence of abdominal obesity was estimated at 33.6% (95% CI: 32.5 to 34.6%). In Peru, altitude was significantly and inversely associated with abdominal obesity, decreasing with higher altitudes: 1500-2999 meters above mean sea level (MAMSL) vs <1500 MAMSL, adjusted prevalence rate [aPR]= 0.86 (95% CI: 0.75 to 0.97); ≥3000 MAMSL vs <1500 MAMSL, aPR= 0.98 (95% CI: 0.87 to 1.11), when adjusting by age, gender and residence area (rural/urban). However, this association was significantly modified by age and gender (p< 0.001). Conclusion: Abdominal obesity is highly prevalent in Peru and decreases significantly with altitude, but age and gender modify this association. Thus, abdominal obesity appears to affect older women from low altitudes more than younger men from high altitudes.", "keywords": [ "Abdominal obesity", "Altitude", "Obesity", "Waist Circumference", "Peru" ], "content": "Introduction\n\nThe increasing prevalence of obesity represents a significant public health problem across low- to high-income countries1. The main reason is that obesity is strongly associated with morbidity and mortality, mostly due to type 2 diabetes, cancer and cardiovascular diseases2. However, body fat distribution, particularly that of abdominal obesity, has been reported as a better predictor of overall morbidity and mortality than total adiposity or obesity defined by body mass index (BMI)3,4. Furthermore, abdominal obesity is difficult to diagnose in routine clinical care because it requires access to computed tomography5 or magnetic resonance imaging6 for precise quantification. Thus, the most commonly used surrogate to diagnose abdominal obesity in clinical care and research examinations is waist circumference7,8. This surrogate has proven to perform well at sea level; however, little is known about its usefulness in high altitudes.\n\nIn Peru, as in most Latin-American countries, the prevalence of obesity among children, adolescents and adults have grown consistently in recent decades. Among Peruvian adults, estimates of the national prevalence of obesity have grown from approximately 9% in 1975 to 21% in 20179. However, this prevalence seems to vary substantially by altitude.\n\nEpidemiological studies carried out in the United States10 and Peru11 among adults and children12 have described an inverse association between altitude and obesity. A previous study reported that the risk of obesity in Peru decreases by approximately 26% at between 1500–2999 meters above mean sea level (MAMSL), and by 46% at over 3000 MAMSL, as compared to at 0–499 MAMSL11.\n\nConsequently, this study further assesses the association between altitude and abdominal obesity, when adjusted by standard sociodemographic variables. Additionally, we plan to estimate the prevalence of abdominal obesity by different cutoffs.\n\n\nMethods\n\nThe study employed a cross-sectional multicentric study design. Data were accessed from the Peruvian National Household Survey (ENAHO), undertaken annually by the Peruvian National Institute of Statistics and Information (INEI) and the National Center for Food and Nutrition (CENAN) to assess social living conditions. For this purpose, the INEI and CENAN surveyed a representative sample of the Peruvian population using a probabilistic, stratified, multi-stage design, independent for each region, to collect data on participants of ≥2 months of age13. ENAHO survey eligibility criteria were inhabitants of Peruvian households, including family members, non-family members and domestic workers (with or without payment) that cohabitated during the 30 days prior to the survey, excluding households of 10 or more inhabitants13. In this study, we used ENAHO 2013 data to assess the prevalence of abdominal obesity and its association with altitude, while adjusting for their primary demographics and design effect.\n\nThe study outcome was abdominal obesity: we used waist circumference (WC) as a proxy for its diagnosis. During the ENAHO survey, trained personnel measured the subject’s WC at the vertical position of the midpoint between the lowest rib and the border of the iliac crest8. We interpreted this measurement by using the cutoffs proposed by Adult Treatment Panel III guidelines (ATP III) for abdominal obesity: WC >102 cm for men and >88 cm for women14,15. Additionally, for comparison, we assessed the cutoffs proposed by the Latin-American Diabetes Association (ALAD): WC ≥94 cm for men and ≥88 cm for women16 and that of the International Diabetes Federation (IDF): WC >90 cm for men and >80 cm for women17. Furthermore, we define abdominal obesity as a weight to height ratio (WtHR) ≥0.518 and obesity as a BMI ≥30 kg/m2.\n\nTo facilitate comparisons and interpretability, we categorized altitude as low (<1500 MAMSL), moderate (1500–2999 MAMSL), and high (≥3000 MAMSL). Likewise, individuals were categorized by age as young adults (20–39 years), adults (40–59 years) and elders (≥60 years). We classified the area of residence as urban or rural based on the rural index of each district, which is assessed annually by INEI. Nutritional status was assessed by BMI and categorized using WHO standard cutoffs as underweight (<18.5 kg/m2), normal (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2), and obese (≥30 kg/m2)19.\n\nWe estimated the prevalence of abdominal obesity by considering survey sampling weights by using STATA survey (svy) commands and excluding registers with missing study outcomes. We assessed bivariate association using Spearman’s rank-order correlation. Considering that the prevalence of abdominal obesity in Peru is not rare9, we estimated the adjusted prevalence ratio as a measure of association instead of the odds ratio20. Thus, we used a log-binomial regression model that has robust variance, rather than a Poisson regression model, to adjust our prevalence ratio estimates by gender, age group and area of residence21. Finally, we tested whether sex and age modified the association between abdominal obesity and altitude using the Wald test. All statistical analyses were performed using STATA/MP 14.0 for Mac (StataCorp LP, College Station, TX), and the results of statistical tests were interpreted and summarized with 95% confidence intervals.\n\nAccording to the Regulation of Ethics in Research of the Peruvian National Institute of Health, this study did not require approval or exemption from an ethics committee because the database is publicly available.\n\n\nResults\n\nWe analyzed a population sample of 20,489 subjects from 703 different locations across 25 administrative regions of Peru. To summarize population demographics, most subjects were either female (51.6%), adults between 20 to 39 years of age (39.8%), or inhabitants from urban areas (79.6%)22. Of these three demographic measures, both age groups (p=0.0006) and area of residence (p<0.0001) distribution varied significantly by altitude (Table 1).\n\nParameters estimated considering the design effect and the complexities of the survey; WtHR, waist-to-height ratio; MAMSL, meters above mean sea level; ATP III, Adult Treatment Panel III guidelines; ALAD, Latin-American Diabetes Association; IDF, International Diabetes Federation.\n\nThe prevalence of abdominal obesity in Peru was 33.6% (95% CI: 32.5% - 34.6%) when using WC and the ATP III cutoff, 44.4% (95% CI: 43.2% - 45.6%) using the ALAD cutoff and 64.1% (95% CI: 63.0% - 65.2%) using the IDF cutoff (Table 1). Regardless of the cutoff used (i.e. ATP III, ALAD, or IDF), the prevalence of abdominal obesity decreased significantly (p<0.001) with altitude: abdominal obesity was more prevalent at low elevations (<1500 MAMSL), less prevalent at moderate elevations (1500–2999 MAMSL), and lowest at high elevations (≥3000 MAMSL) (Table 1 and Figure 1).\n\nATP III, Adult Treatment Panel III guidelines; ALAD, Latin-American Diabetes Association; IDF, International Diabetes Federation; MAMSL, meters above mean sea level.\n\nSimilarly, we estimated the prevalence of abdominal obesity in Peru using waist to height ratio (WtHR) to be 83.9% (95% CI: 83.1%-84.6%). Like the previous model that employed WC as a surrogate measure of abdominal obesity, the present model also demonstrated an inverse association between abdominal obesity and altitude category. In this model, the prevalence of abdominal obesity (as defined by WtHR) was 86.1% (95% CI: 85.1%-87.1%) for those at low altitudes, 80.7% (95% CI: 78.9%-82.7%) at moderate altitudes, and 77.9% (95% CI: 76.1% to 79.6%) at high altitudes (p<0.001) (Table 1).\n\nWe estimated the total prevalence of obesity in Peru by BMI to be 19.7% (95% CI: 18.9%-20.6%). Like that of abdominal obesity, the prevalence of obesity was inversely related to the categories of altitude that we defined. Obesity prevalence was 22.9% (95% CI: 21.7%-24.1%) at low elevations, 15.0% (95% CI: 13.5%-16.6%) at moderate elevations, and 11.8% (95% CI: 10.6%-13.1%) at high elevations for those living at or over 3000 MAMSL, respectively (p<0.001) (Table 1).\n\nEstimates of abdominal obesity prevalence vary significantly with altitude and in models that use different standard diagnostic cutoffs. When comparing the estimated prevalence of abdominal obesity using ATP III, ALAD and IDF cutoffs (Table 1 and Figure 1), there were significant differences between them (p<0.001 at each paired comparison). The same variability was observed regardless of age group, gender, and residence area (Table 2). Furthermore, in the correlation analysis (Table 3), we found that using the ATP III cutoff resulted in a stronger correlation with obesity (Spearman´s ρ = 0.55; p<0.001), as compared with the ALAD (Spearman´s ρ = 0.53; p<0.001) and IDF cutoffs (Spearman´s ρ = 0.37; p<0.001). However, the ATP III cutoff also has a weaker correlation with altitude (Spearman´s ρ = 0.12; p<0.001). Additionally, we found that the prevalence of abdominal obesity, as defined by WtHR >0.5, has only a moderate correlation with the prevalence of obesity (Spearman´s ρ = 0.43; p<0.001) and a weak correlation with altitude (Table 3).\n\nEstimates considering the design effect and the complexities of the survey; abdominal obesity estimated using the cutoffs proposed by Adult Treatment Panel III guidelines (ATP III); MAMSL, meters above mean sea level; ALAD, Latin-American Diabetes Association; IDF, International Diabetes Federation.\n\nAll the correlations estimated in the table resulted in a p-value <0.001 when tested as equal to zero. BMI, body mass index; AO, abdominal obesity; WtHR, waist to height ratio; ATP III, Adult Treatment Panel III guidelines; ALAD, Latin-American Diabetes Association; IDF, International Diabetes Federation.\n\nThe prevalence of abdominal obesity and obesity vary significantly by altitude in Peru and are inversely associated with altitude category (trend analysis p<0.001 for both), regardless of age group, gender and residence area (Table 4). Both abdominal obesity and obesity prevalence were significantly higher among females than males (p<0.001 for both) and across rural areas than in urban areas (p<0.001 for both). The prevalence of obesity and abdominal obesity were significantly lower among young adults (20–39 years) than among adults (40–59 years); however, both obesity and abdominal obesity prevalence were significantly higher in young adults than elders (≥60 years old).\n\n*Estimates considering the design effect and the complexities of the survey; +, abdominal obesity estimated using the cutoffs proposed by Adult Treatment Panel III guidelines (ATP III); MAMSL, meters above mean sea level; BMI, body mass index; 95% CI, confidence intervals of 95%.\n\nRegression analyses demonstrated that the prevalence of abdominal obesity was significantly associated with altitude when either unadjusted and adjusted by age groups, gender, and residence. Additionally, we observed significant effect modification of this association by age group and gender, which seems to be particularly high at altitudes over 3000 MAMSL. Once adjusted by the interaction terms, the association between abdominal obesity and altitude varies significantly by gender, age group and residence area, with different patterns of distribution at different altitudes. At lower altitudes (<1500 MAMSL), the prevalence of abdominal obesity exhibits a positive trend increasing by age group, while above 1500 MAMSL, it exhibits an inverted-u shaped relationship (Figure 2).\n\nThe association between abdominal obesity and altitude vary greatly by gender and age group, which behave as effect modifiers.\n\nIn the regression analysis, we found that altitude, age groups, gender, and residential area were significantly associated with the prevalence of abdominal obesity in Peru (Table 5). Based on our multivariate regression analysis outputs, we observed that the prevalence of abdominal obesity decreased with altitude, increased with age, and is lower among male and rural populations. However, contrary to what was observed for the prevalence of abdominal obesity by altitude in the case of gender and residence area, both of which decrease with altitude, the variability of the prevalence of abdominal obesity by age group exhibits different patterns of distribution at different altitudes. Overall, the prevalence of abdominal obesity in Peru is higher among women ≥60 years living at <1500 MAMSL (68.4%; 95% CI, 64.6 to 71.9), and lower among men between 20 to 39 years of age living al ≥3000 MAMSL (2.8%; 95% CI, 1.6 to 4.8), exhibiting an inverted-u shaped relationship (Figure 2).\n\nDesign effect was considered according to complex survey data; ATP III, Adult Treatment Panel III guidelines; PR, prevalence rates; CI 95%, confidence intervals of 95%, MAMSL: meters above mean sea level; °, non-significant p-value; †, p-value <0.05; ‡, p-value <0.001.\n\n\nDiscussion\n\nThe prevalence of abdominal obesity in Peru is high and decreases with altitude, an association that is modified by age and gender. This prevalence was higher among women over 60 years of age below 1500 MAMSL, and lowermost among men 20 to 39 years of age over 3000 MAMSL, exhibiting an inverted-u shaped relationship. Understanding the intricacies of this association is critical in countries with high elevation such as Peru, where approximately 20% of the Peruvian population lives at or above 3000 MAMSL23.\n\nThe usefulness of WC as an indicator of abdominal obesity is quite clear; however, there is a permanent discussion regarding the cutoffs for its diagnosis. WC varies by ethnic groups, which has generated the recommendation that each country or region produces its cutoffs24. Worldwide, the most used cutoffs for WC are the ones proposed by the ATP III, which are primarily specific for adult European Caucasian populations14,15.\n\nThere are some efforts in Latin America to propose WC cutoffs for their population. A recent study carried out in five Latin American countries recommended using cutoffs of 90–92 cm for women and 94 cm for men25. In Peru, the PREVENTION study proposed WC cutoffs at high altitude (~2600 MAMSL) of 87 cm for women and 97 cm for men based on abnormalities of intima-media thickness and cardiovascular manifestations26. Similarly, different countries have proposed their cutoffs for WC, including Portugal (91 and 97 cm)27, China (80 and 84 cm)28, and South Asian countries (84 and 88 cm)29. In our study, different cutoffs produced a wide range of estimates for the prevalence of abdominal obesity. We observed that when using ATP III cutoffs, the estimated prevalence of abdominal obesity was over three times higher among women than in men (51% vs 15%).\n\nFurthermore, regardless of altitude, these differences seem to be even larger ≥3000 MAMSL (40% vs 7%). These differences are similar to those reported previously30, so we believe they can be explained by both the altitude effect and the cutoffs itself, which are gender-differentiated. Further studies are needed to assess the necessity of specific cutoffs corrected by altitude, gender, and age.\n\nAnother important finding of our study is that the prevalence of abdominal obesity varies significantly between urban and rural areas, a difference that remains consistent at different altitudes. As reported elsewhere, the prevalence of abdominal obesity in Peru is higher in urban areas than in rural areas31. However, such a difference between urban and rural areas seems to increase with higher altitudes, ranging from 1.7:1 at <1500 MAMSL to 2.1:1 at ≥3000 MAMSL. This finding is relevant in countries with large populations living over 3000 MAMSL, due to the cardiovascular risk that this could imply.\n\nRegardless of WC cutoffs utilized, the mean WC in the Peruvian population living at high altitudes is high. In our study, at >3000 MAMSL the mean WC among men was 87.1 cm and among women 86.0 cm, which are lower than those reported at ~3600 MAMSL in La Paz-Bolivia (93 cm in women and 93 cm in men)32 and close to those reported at ~3660 MAMSL in Tibet (84.5 cm overall)33.\n\nAccording to our results, by both WC and WtHR, Peruvians who live at higher altitudes have a lower prevalence of abdominal obesity than those living a lower altitude. This finding concurs with previous reports11,34; moreover, a higher percentage of overweight (36.3% vs 25.3%), obesity (17.5% vs 8.5%), hypercholesterolemia (18.9% vs 14.6%), low HDL (45.7% vs 40.3%), hypertension (9.8% vs 3.9%) and glycemia >126 mg/dL (2.9% vs 0.9%) were observed in people living above 3000 MAMSL vs below 1000 MAMSL34. Overall, the lower cardiovascular risk observed at higher altitudes could be explained in part by the lower levels of urbanization and income, commonly reported in developing countries35. Also, it might be explained by the variability in the progress of the epidemiological transition in Peru observed at different altitudes36. It is important to highlight that a WtHR >0.5 seems to overestimate Peruvian abdominal obesity. Regardless of the evidence18, if we use a cutoff of 0.5, over 80% of the Peruvian population is classified as having abdominal obesity. Further studies are needed to assess the usefulness of such an indicator in Latin-American countries such as Peru.\n\nWe should mention as a limitation that the ENAHO survey was meant to represent Peru’s nutritional status, and the sample might not represent all altitudes of the country. Likewise, it is essential to emphasize that Peru is one of the few countries with many large populations over 3000 MAMSL. Therefore, the association between altitude and obesity could remain unnoticed at low altitude countries. Another limitation is the absence of variables such as socioeconomic status, education level, physical activity and diet. However, the area of residence (urban and rural) is a variable that encompasses socioeconomic and educational aspects in our country.\n\nIn conclusion, our study found that abdominal obesity is highly prevalent in Peru and that abdominal obesity varies substantially by altitude, age, gender, and urbanization. Overall, the prevalence of abdominal obesity decreases with altitude, but age and gender modify such association; abdominal obesity seems to affect older women from low altitudes more than younger men from high altitudes. These findings should help to guide interventions to reduce Peruvian’s cardiovascular risk, which should be a matter of more significant concern in future years.\n\n\nData availability\n\nFigshare: Altitude and its inverse association with abdominal obesity in an Andean country. https://doi.org/10.6084/m9.figshare.9920234.v122\n\nThis project contains the following underlying data:\n\n- altitude_abdominal_obesity_dataset.xls (demographic and abdominal obesity data for participants)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nGBD 2015 Obesity Collaborators, Afshin A, Forouzanfar MH, et al.: Health Effects of Overweight and Obesity in 195 Countries over 25 Years. N Engl J Med. 2017; 377(1): 13–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuh DP, Zhang W, Bansback N, et al.: The incidence of co-morbidities related to obesity and overweight: a systematic review and meta-analysis. BMC Public Health. 2009; 9: 88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYusuf S, Hawken S, Ounpuu S, et al.: Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. Lancet. 2004; 364(9438): 937–952. PubMed Abstract \| Publisher Full Text\n\nHoefle G, Saely CH, Aczel S, et al.: Impact of total and central obesity on vascular mortality in patients undergoing coronary angiography. Int J Obes (Lond). 2005; 29(7): 785–791. PubMed Abstract \| Publisher Full Text\n\nSeidell JC, Björntorp P, Sjöström L, et al.: Regional distribution of muscle and fat mass in men--new insight into the risk of abdominal obesity using computed tomography. Int J Obes. 1989; 13(3): 289–303. PubMed Abstract\n\nRoss R, Shaw KD, Martel Y, et al.: Adipose tissue distribution measured by magnetic resonance imaging in obese women. Am J Clin Nutr. 1993; 57(4): 470–475. PubMed Abstract \| Publisher Full Text\n\nde Koning L, Merchant AT, Pogue J, et al.: Waist circumference and waist-to-hip ratio as predictors of cardiovascular events: meta-regression analysis of prospective studies. Eur Heart J. 2007; 28(7): 850–856. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Waist Circumference and Waist-Hip Ratio: Report of a WHO Expert Consultation, Geneva, 8-11 December 2008. World Health Organization; 2011. Reference Source\n\nPajuelo Ramírez J, Ramírez JP: La obesidad en el Perú. Anales de la Facultad de Medicina. 2017; 78(2): 73. Publisher Full Text\n\nVoss JD, Masuoka P, Webber BJ, et al.: Association of elevation, urbanization and ambient temperature with obesity prevalence in the United States. Int J Obes (Lond). 2013; 37(10): 1407–12. PubMed Abstract \| Publisher Full Text\n\nWoolcott OO, Gutierrez C, Castillo OA, et al.: Inverse association between altitude and obesity: A prevalence study among andean and low-altitude adult individuals of Peru. Obesity (Silver Spring). 2016; 24(4): 929–37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPajuelo Ramírez J, Miranda Cuadros M, Bernui Leo I: Asociación entre altitud de residencia y malnutrición en niños peruanos menores de cinco años [Association between altitude of residence and malnutrition in Peruvian children under five years of age]. Acta Med Peru. 2017; 34(4): 259–265. Reference Source\n\nInstituto Nacional de Estadística e Informática (INEI): Peru - Encuesta Nacional de Hogares Sobre Condiciones de Vida y Pobreza 2017. [National Institute of Statistics and Informatics (INEI). Peru - National Household Survey on Living Conditions and Poverty 2017.] Reference Source\n\nExpert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults: Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III). JAMA. 2001; 285(19): 2486–2497. PubMed Abstract \| Publisher Full Text\n\nHan TS, van Leer EM, Seidell JC, et al.: Waist circumference action levels in the identification of cardiovascular risk factors: prevalence study in a random sample. BMJ. 1995; 311(7017): 1401–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAsociación Latinoamericana de Diabetes (ALAD): Epidemiología, Diagnóstico, Control, Prevención y Tratamiento del Síndrome Metabólico en Adultos. [Latin American Diabetes Association (ALAD). Epidemiology, Diagnosis, Control, Prevention and Treatment of Metabolic Syndrome in Adults]. Rev Asoc Latinoam Diab. 2010; 18(1): 25–44. Reference Source\n\nAlberti KG, Zimmet P, Shaw J: Metabolic syndrome--a new world-wide definition. A Consensus Statement from the International Diabetes Federation. Diabet Med. 2006; 23(5): 469–480. PubMed Abstract \| Publisher Full Text\n\nBrowning LM, Hsieh SD, Ashwell M: A systematic review of waist-to-height ratio as a screening tool for the prediction of cardiovascular disease and diabetes: 0·5 could be a suitable global boundary value. Nutr Res Rev. 2010; 23(2): 247–269. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Obesity: preventing and managing the global epidemic. Report of a WHO consultation. 2000. Reference Source\n\nTajeu GS, Sen B, Allison DB, et al.: Misuse of odds ratios in obesity literature: an empirical analysis of published studies. Obesity (Silver Spring). 2012; 20(8): 1726–1731. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEspelt A, Marí-Dell'Olmo M, Penelo E, et al.: Applied Prevalence Ratio estimation with different Regression models: An example from a cross-national study on substance use research. Adicciones. 2017; 29(2): 105–112. PubMed Abstract \| Publisher Full Text\n\nPajuelo-Ramírez J, Torres-Aparcana H, Agüero-Zamora R, et al.: Altitude and its inverse association with abdominal obesity in an Andean country: Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9920234\n\nInstituto Nacional de Estadística e Informática: Peru - Resultados definitivos de los Censos Nacionales 2017: XII de Población, VII de Vivienda y III de Comunidades Indígenas. [National Institute of Statistics and Informatics. Peru - Final Results of the 2017 National Censuses: XII Population, VII Housing and III Indigenous Communities]. 2018. Reference Source\n\nMisra A, Wasir JS, Vikram NK: Waist circumference criteria for the diagnosis of abdominal obesity are not applicable uniformly to all populations and ethnic groups. Nutrition. 2005; 21(9): 969–976. PubMed Abstract \| Publisher Full Text\n\nAschner P, Buendía R, Brajkovich I, et al.: Determination of the cutoff point for waist circumference that establishes the presence of abdominal obesity in Latin American men and women. Diabetes Res Clin Pract. 2011; 93(2): 243–247. PubMed Abstract \| Publisher Full Text\n\nMedina-Lezama J, Pastorius CA, Zea-Diaz H, et al.: Optimal definitions for abdominal obesity and the metabolic syndrome in Andean Hispanics: the PREVENCION study. Diabetes Care. 2010; 33(6): 1385–1388. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRaposo L, Severo M, Santos AC: Adiposity cut-off points for cardiovascular disease and diabetes risk in the Portuguese population: The PORMETS study.Tauler P, ed. PLoS One. 2018; 13(1): e0191641. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang H, Liu A, Zhao T, et al.: Comparison of anthropometric indices for predicting the risk of metabolic syndrome and its components in Chinese adults: a prospective, longitudinal study. BMJ Open. 2017; 7(9): e016062. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatel SA, Deepa M, Shivashankar R, et al.: Comparison of multiple obesity indices for cardiovascular disease risk classification in South Asian adults: The CARRS Study. Kiechl S, ed. PLoS One. 2017; 12(4): e0174251. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPajuelo J, Sánchez-Abanto J, Torres HL, et al.: Prevalencia del síndrome metabólico en pobladores peruanos por debajo de 1 000 y por encima de los 3 000 msnm [Prevalence of the metabolic syndrome in Peruvian settlers below 1,000 and above 3,000 meters above sea level]. Anales de la Facultad de Medicina. 2012; 73(2): 101–106. Publisher Full Text\n\nCarrillo-Larco RM, Bernabé-Ortiz A, Pillay TD, et al.: Obesity risk in rural, urban and rural-to-urban migrants: prospective results of the PERU MIGRANT study. Int J Obes (Lond). 2016; 40(1): 181–185. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaye-Huanca EO, Navia-Bueno M de P: Prevalencia y factores de riesgo asociados para sobrepeso y obesidad en la población adulta de la ciudad de La Paz [Prevalence and associated risk factors for overweight and obesity in the adult population of the city of La Paz]- Gestión 2014. Cuad Hosp Clin. 2018; 59: 31–40. Reference Source\n\nSherpa LY, Deji, Stigum H, et al.: Obesity in Tibetans aged 30-70 living at different altitudes under the north and south faces of Mt. Everest. Int J Environ Res Public Health. 2010; 7(4): 1670–1680. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPajuelo-Ramírez J, Sánchez-Abanto J, Arbañil-Huamán H: Las enfermedades crónicas no transmisibles en el Perú y su relación con la altitud [Chronic noncommunicable diseases in Peru and their relationship with altitude]. Rev Soc Peru Med Interna. 2010; 23(2): 45–52. Reference Source\n\nSliwa K, Acquah L, Gersh BJ, et al.: Impact of Socioeconomic Status, Ethnicity, and Urbanization on Risk Factor Profiles of Cardiovascular Disease in Africa. Circulation. 2016; 133(12): 1199–1208. PubMed Abstract \| Publisher Full Text\n\nHuicho L, Trelles M, Gonzales F, et al.: Mortality profiles in a country facing epidemiological transition: an analysis of registered data. BMC Public Health. 2009; 9(1): 47. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "55852", "date": "04 Nov 2019", "name": "Antonio Bernabe-Ortiz", "expertise": [ "Reviewer Expertise Noncommunicable diseases (type 2 diabetes", "hypertension and obesity)." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall comment: This paper looks for the association between altitude and abdominal obesity. Although the idea is not novel, extra reports using existing data can help to understand better this. I would expect more details regarding the variables (exposure and outcome) used in the analysis. Moreover, any other kind of models, not that using only three categories for altitude would be more relevant, or verifying if the association is similar for overweight and obesity. This analysis can be improved by looking extra ways to see the association more than the traditional form to check that, for example, using linear regression if possible or polynomic models (quadratic at least).\n\nMajor concerns:\nAbstract: Please change it accordingly to comments below.\nIntroduction:\nObesity has different indicators and in the reference is used similarly for BMI (overall obesity), waist circumference (abdominal obesity), etc. Please be careful and consistent with words used. Some longitudinal studies have been published showing the association of interest and they have not been considered here (e.g. PMID: 294725201). What is the novelty in this paper? Is it altitude? Apparently not as shown in the longitudinal paper... is it the rurality? Usually rural areas are in high altitudes. Is waist circumference an easy measure to do? Is it routinely done? How the usefulness of waist circumference in high altitudes is evaluated in this paper? I think that is no part of this study as pointed out in the last part of the first paragraph. Any reference for the last sentence of the second paragraph? Third paragraph: is the risk of obesity decreasing due to altitude? The reference is a cross sectional study, so is it possible to talk about risk?\nMethods:\nStudy design: why this study is multicentric? How many centers were included? Please explain. If the ENAHO is conducted yearly, why to use the 2013 ENAHO? Why not to use the last one available? Please explain how the sampling was done? Stratified by what? How many stages does the sampling have? Was any criterion related to time living in high altitude (e.g. at least 6 months living in the city/area)? Are 30 days enough to see the potential impact of altitude on health? Why households with 10 or more inhabitants were excluded? Any explanation? What proportion of households in Peru has 10 dwellers or more? Are these decisions biasing results? How altitude was measured? Was GPS used for this? Only a simple calculation of the altitude of the city was used? Please explain. Usually, 2500 meters is used as the cutoff and not 3000... Since this variable is the main exposure, details should be given to understand if any misclassification could be introduced... Only those aged 20 years and more were included in the analysis? How about those between 18 to 20 years or those younger? How the rural index was built? Explain please... Any reference helping to understand this? I am pretty sure the ENAHO stratifies the sample by urban/rural settings... was not the case this time? Categorization of BMI is presented in different way compared to how it was analyzed... please be consistent in definitions used “We assessed bivariate association using...” Association of what? Do you mean correlation between definitions of abdominal obesity? What was the gold standard? “Thus, we used a log-binomial...” Why education level or socioeconomic status were not confounders? As they are associated with obesity and people in high altitude settings, mainly rural, tend to have low SES and low education... should be they confounders? I know these variables are available in the ENAHO survey... why were they excluded? Ethic statement: where the dataset is available... add the link and how to get the data... I know a dataset has been added using Figshare, but that it not all the info available in the ENAHO dataset.\nResults:\nWhat do you mean for “703 different locations”? This part is not explained in the Methods section. Were they the areas with altitude information? Education level and socioeconomic status should be included in the info as they are available in the ENAHO dataset. Waist to height ratio (definition) should be included since the methods section. Was obesity prevalence standardized by age? Authors always said that the prevalence of obesity was lower in high altitude, but that is only the crude estimate... at least age standardization (if possible by sex also) must be conducted to say something like that. “... we found that using the ATP III cutoff resulted in a stronger correlation with obesity”... what kind of obesity? Defined by BMI? By WC? By WtHr? Please use appropriate graphs to see the inverted-u shape relationship... three points are not enough... use appropriate linear regression models? Or used more categories in the exposure variable... for example, an increase every 500 meters... Would be good to know if stratification variables were effect modifiers? What was the test used for this? Although this was defined a priori, it would be good to have these estimates...\nDiscussion:\nI would suggest discussing about the relevance of this paper in the context of other papers (mainly those longitudinal). What is the relevance of the findings? This is not clear as apparently the discussion is used only to compare results with other studies. I would also suggest to tone down some of the sentences as this is only a cross sectional study and not better analysis has been done to improve the understanding of the association between altitude and obesity prevalence.\nMinor concerns:\nCorrect grammar spelling. Paper should be reviewed by an English native speaker.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "56784", "date": "25 Nov 2019", "name": "Ugo Fedeli", "expertise": [ "Reviewer Expertise epidemiology of non-communicable diseases" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is interesting and well written. I have few suggestions:\nMethods, statistical analysis: please add some detail on interaction terms introduced in the log-binomial regression models; also in Results some interpretation about estimates obtained for the altitude x gender and altitude x age group would be useful for the reader.\nPage 6, second paragraph: it is stated that abdominal obesity and obesity prevalence were higher across rural areas than in urban areas; possibly the contrary should be reported.\nTable 5: my advice is to report in three separate columns estimates for unadjusted PR, adjusted PR without interaction terms, and adjusted PR with interaction terms (full model). It is not clear if PR reported in the Abstract are obtained from the full model; my advice is to report in the Abstract PR obtained by the adjusted model without interaction terms.\nIn Discussion it is reported that differences in socio-economic status might be captured by the urban/rural variable. Are there also differences in ethnic background by altitude?\nPage 9, findings are summarized of a previous study reporting a higher percentage of overweight and other cardiovascular risk factors in people living above 3000 m vs. those living below 1000 m. However, these results seem to be in contrast with the present study; please explain.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "56783", "date": "05 Dec 2019", "name": "Yuri Arnold Dominguez", "expertise": [ "Reviewer Expertise Epidemiology of diabetes." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the topic number 1 and referring specifically to what was written in paragraph number one of the Introduction, I consider that not only the abdominal waist is the only predictor of abdominal obesity, there are other indicators as important as that, such as the Waist Index- size (taking into account that the Peruvian population prevails people with short stature), the waist waist index, the conicity index, which are usually more accurate than mere measurement than the abdominal circumference. I recommend that this investigation be continued in a larger sample , that it is even necessary to evaluate risk factors such as physical activity and diet, which are major confounders.\nOtherwise I catalogue the article as excellent.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1738
https://f1000research.com/articles/8-2142/v1	24 Dec 19	{ "type": "Systematic Review", "title": "Humanistic burden and economic impact of chronic kidney disease: a systematic literature review", "authors": [ "Caroline Freeman", "Lucia Giles", "Polly Field", "Elisabeth Sörstadius", "Heleen van Haalen", "Lucia Giles", "Polly Field", "Elisabeth Sörstadius", "Heleen van Haalen" ], "abstract": "Background: Chronic kidney disease (CKD) is increasing in prevalence worldwide. Progression of CKD to end-stage renal disease (ESRD) can result in the requirement for renal replacement therapy, which incurs considerable healthcare costs and imposes restrictions on patients’ daily living. This systematic review was conducted to inform understanding of the humanistic and economic burden of CKD by collecting quality of life (QoL), symptom burden, and cost and resource use data, with a focus on the impact of disease progression. Methods: Embase, MEDLINE, the Cochrane Library, and conference proceedings were searched in May 2017 according to predefined inclusion criteria. Data were extracted for full publications reporting either QoL or symptom burden (published 2007–2017; reporting data from ≥ 100 patients) or costs and resource use (published 2012–2017). Relevant QoL studies were those that used the 6-dimension or 8-, 12-, or 36-item Short-Form Health Surveys, 5-dimension EuroQol questionnaire, Healthy Days/Health-Related Quality of Life questionnaire, or Kidney Disease Quality of Life Questionnaire. Results: Data were extracted from 95 studies reporting QoL data, 47 studies reporting cost and resource use data, and eight studies reporting descriptions of symptoms; 12 studies (seven QoL; five costs/resource use) reported data for patients with and without CKD, and 15 studies (seven QoL; eight costs/resource use) reported data by disease stage. Patients with CKD, including those with ESRD, had worse QoL than those with normal kidney function, and incurred higher healthcare costs. Disease progression was associated with cost increases, particularly for later stages and in patients receiving dialysis. Increasing CKD severity was also associated with reductions in QoL, although not all studies identified showed a consistent decrease with increasing disease stage. Conclusions: The presence of CKD and CKD progression are associated with reductions in patients’ QoL and increased economic impact. This may be mitigated by interventions that slow progression.", "keywords": [ "chronic kidney disease", "end-stage renal disease", "quality of life", "healthcare costs", "disease severity", "systematic review", "humanistic burden", "economic burden" ], "content": "Introduction\n\nChronic kidney disease (CKD) is characterized by a gradual loss of kidney function over time. With an estimated global prevalence of 11–13%, CKD is a common condition that is associated with a significant economic burden across the world1. The prevalence of the disease is rising, owing in part to an increase in the median age of populations worldwide, and the growing number of individuals with diabetes mellitus (DM) or hypertension1. These conditions are the two main causes of CKD and are commonly present in patients with diminished renal function2. In the USA, for example, approximately 40% of people with CKD also have DM, and 32% of people with CKD have hypertension3.\n\nWhen CKD progresses, patients may experience complications such as anaemia, cardiovascular disease (CVD), peripheral arterial disease, pruritus, and increased risk of infection. Both disease progression and its associated complications require medical treatment, which further impacts patients’ quality of life (QoL) and contributes to the humanistic and economic burden of CKD4,5. Moreover, progression to end-stage renal disease (ESRD) has a significant effect on patients’ daily lives and is often associated with considerable costs due to the common requirement for renal replacement therapy (RRT) via dialysis or kidney transplantation1. Therefore, slowing the rate of progression of CKD to advanced stages, in particular to ESRD, is an important medical objective4.\n\nMany studies have investigated the humanistic and economic burden of CKD, although quantification of this remains challenging owing to differences between methodologies and patient populations across studies. To gain a better understanding of the current evidence, and evidence gaps, we conducted systematic reviews (SRs) to identify relevant evidence on the humanistic burden of CKD, defined here as the effect of CKD on patients’ QoL, and the economic burden, comprising resource use and healthcare costs associated with CKD. In particular, we reviewed data on QoL and costs for patients with CKD compared with the general population, and across disease stages.\n\n\nMethods\n\nTwo systematic searches, one on humanistic burden/QoL and one on economic burden, were performed in MEDLINE and MEDLINE In-Process, Embase, and the Cochrane Library via Ovid. Cut-off dates were January 2002 and May 2017 for the humanistic burden SR and January 2007 and May 2017 for the economic burden SR; the shorter review window for the economic burden SR was specified because economic data are considered to decrease in relevance more quickly than QoL data. As supplementary searches, congress abstracts published between 2015 and 2017 (or the most recent 2 years available) from relevant health economics and outcomes research and nephrology meetings were reviewed. The search strings used to identify evidence are listed in Extended data: Supplementary content 16.\n\nAbstracts and titles identified were screened by an independent reviewer to determine whether they met the PICOS (patient, interventions, comparisons, outcomes, and study design) eligibility criteria (Table 1), in accordance with 2009 Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines7. All publications that met entry criteria for review were obtained as full articles and reassessed against the predefined inclusion criteria. Owing to the large number of citations meeting these criteria, a decision was taken to restrict the extraction of data from eligible studies. For both SRs, data were extracted only from full publications. This meant that, although congress abstract screening was performed, no data from congress abstracts were extracted. For the humanistic burden SR, study publication dates for data extraction were restricted to 2007–2017; for the economic burden SR, the relevant time period was restricted to 2012–2017. Furthermore, for the humanistic burden SR, data were not extracted if the study population included fewer than 100 patients with CKD or if the study did not use any of the following instruments:\n\n36-item Short-Form Health Survey (SF-36)\n\n12-item Short-Form Health Survey (SF-12)\n\n8-item Short-Form Health Survey (SF-8)\n\n6-dimension Short-Form Health Survey (SF-6D)\n\n5-dimension EuroQol questionnaire (EQ-5D)\n\nHealthy Days/Health-Related Quality of Life (HRQoL) questionnaire\n\nKidney Disease Quality of Life Questionnaire (KDQoL).\n\nCKD, chronic kidney disease. eGFR, estimated glomerular filtration rate. ESRD, end-stage renal disease. HSUV, health state utility value. PRO, patient-reported outcome. QoL, quality of life. SR, systematic review.\n\nFor each publication, information was extracted into a data extraction table. Studies were listed according to inclusion of QoL, symptom burden or cost, and resource use data. For each study, patients’ CKD stage, RRT status [pre-dialysis, dialysis (including dialysis modality) or renal transplant], age, and comorbidities including CVD and the presence of DM were recorded. No risk of bias assessment between or within studies was performed.\n\nThese SRs were conducted to collect information on the overall impact of CKD development and progression on patients’ QoL or their healthcare costs. Therefore, we chose to focus this review on studies that compared QoL or costs for patients with CKD and individuals without CKD, or that compared by CKD severity, defined by either disease stage or estimated glomerular filtration rate (eGFR) category. Other studies identified in the SRs are grouped into key themes and listed separately to indicate the scope of the evidence identified in our searches.\n\n\nResults\n\nIn total, 5219 papers were identified in the initial searches, of which 1114 papers were removed as duplicates, and 4105 were included for screening by abstract and title. This screening identified 3539 papers that did not meet the inclusion criteria. In total, 444 papers were included for full paper review. Following full paper review, 284 references were identified for inclusion, plus 79 abstracts identified in supplementary searches. In total, data were extracted from 95 references reporting QoL data, 47 references reporting cost and resource use data, and eight references reporting descriptions of symptoms. A PRISMA flow diagram is shown in Figure 1. Details of the patient populations in the studies discussed in this review are given in Table 2. Figure 2 shows all studies identified in these SRs, grouped into key themes according to the data reported.\n\naThe congresses included in this search were the International Society for Pharmacoeconomics and Outcomes Research US and European congresses, the European Renal Association–European Dialysis and Transplant Association congress, the World Congress of Nephrology, the American Society of Nephrology Kidney Week, and the National Kidney Foundation Spring Clinical Meeting. PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses. QoL, quality of life. SR, systematic review.\n\nCKD, chronic kidney disease. DM, diabetes mellitus. eGFR, estimated glomerular filtration rate. EQ-5D, 5-dimension EuroQol questionnaire. ESA, erythropoietin-stimulating agent. ESRD, end-stage renal disease. HD, haemodialysis. HRQoL-4, 4-item Healthy Days/Health-Related Quality of Life questionnaire. KDQoL, Kidney Disease Quality of Life questionnaire. LoS, length of stay. MCS, mental component summary. NR, not reported. PCS, physical component summary. PD, peritoneal dialysis. SF-6D, 6-dimension Short-Form Health Survey. SF-8/-12/-36, 8-/12-/36-item Short-Form Health Survey. aTaken as those reported as in the ESRD/dialysis category. b14.4% of patients at CKD stage 4 in this study were receiving dialysis. c70.3% of patients at CKD stage 5 in this study were receiving dialysis.\n\naSome studies fell into more than one category. CKD, chronic kidney disease. HRQoL, health-related quality of life.\n\nSeveral studies compared the QoL of patients with CKD with that of individuals in the general population. These studies reported overall measures associated with QoL, as well as physical component summary (PCS), mental component summary (MCS), and individual domain scores from the SF-36.\n\nAcross the studies identified, patients with CKD experienced lower physical and mental QoL compared with the general population. A study by Davison et al. that measured QoL using the SF-6D found that a mixed population of patients with CKD who were either pre-dialysis (CKD stage 3–4, anticipated to require dialysis within 12 months; 34%) or dialysis-dependent (66%) reported lower QoL than an age-matched subset of the general population (SF-6D score: 0.67 vs 0.79)8. A study by Nguyen et al. found that patients with pre-dialysis CKD (stages 3–5), the majority (69.1%) of whom had CKD stage 3a, spent, on average, a greater number of days over a 30-day period inactive owing to poor physical and mental health (3.1 vs 1.5 days; P < 0.001) and a greater number of days with poor physical health (5.1 vs 3.0 days; P < 0.01) compared with a sample of the US general population9. However, a study conducted by Agarwal et al. in the USA, which measured only sleep quality, found no difference between patients with pre-dialysis CKD or dialysis-dependent ESRD and a matched population without CKD10.\n\nCompared with the general population, patients receiving dialysis experienced lower QoL. In total, four studies reported SF-12/SF-36 scores in patients receiving dialysis compared with the general population11–14. Studies by Wan et al. and Wang et al. reported a statistically significant reduction in SF-12 and SF-36 PCS scores (9.9% and 37.6%, reductions, respectively; both P < 0.001; Figure 3)12,13. Boini et al. also reported a reduction in SF-12 PCS scores in patients undergoing dialysis compared with the general population of the USA and France (21.0% and 21.5%, respectively; Figure 3); however, no statistical analysis was reported11. Boini et al.11 and Wang et al.13 reported a decrease in SF-12 (USA: 14.2%; France: 9.1%; P = NR) and SF-36 (23.0%; P < 0.001) MCS scores, respectively, in patients who were dialysis-dependent compared with the general population, whereas Wan et al.12 reported a statistically significant increase in SF-12 MCS scores (9.8%; P < 0.001) in patients receiving dialysis compared with the general population of Hong Kong (Figure 3).\n\naMH, P < 0.01 versus GP; RL-P and BP, P < 0.005 versus GP; PCS, MCS, PF, and GHP, P < 0.001 versus GP. bFor all domains, P < 0.001 versus average. cPatients with ESRD versus GP (age- and sex-adjusted): BP, P < 0.05 versus GP; MH, P < 0.005 versus GP; PF, RL-P, GHP, V, SF, RL-M, P < 0.001 versus GP. Patients on palliative care versus GP (age- and sex-adjusted): RL-P and V, P < 0.05 versus GP; PF and SF, P < 0.001 versus GP. dNo statistical analysis reported. eFor the general population of the USA, MCS and PCS scores were both reported as 50. BP, bodily pain, CKD, chronic kidney disease. ESRD, end-stage renal disease. GP, general population. HD, haemodialysis. MH, mental health. PF, physical functioning. RL-M, role-limiting (mental). RL-P, role-limiting (physical). SF, social functioning. SF-12/36, 12-/36-item Short-Form Health Survey. V, vitality.\n\nThe studies by Wan et al., Wang et al., and Yong et al. reported individual SF-12 and SF-36 domain scores for patients undergoing dialysis compared with the general population12–14. Wan et al. found that patients receiving dialysis reported significantly higher scores for the mental health domain (7.0%; P = 0.006) but lower scores in the physical functioning (20.3%; P < 0.001), role-limiting (physical; 9.8%; P = 0.004), bodily pain (10.0%; P = 0.004), and general health (20.0%; P < 0.001) domains than those without CKD (Figure 3)12. In studies by Wang et al. and Yong et al., a statistically significant reduction in scores across all domains of the SF-36 (P < 0.001 and P < 0.05, respectively) was reported in patients receiving dialysis compared with the general population (Figure 3)13,14. Yong et al. also reported lower SF-36 domain scores in patients with ESRD undergoing palliative care compared with the general population. A statistically significant difference was found only in the physical functioning (P < 0.001), role-limiting (physical; P = 0.027), social functioning (P < 0.001), and vitality (P = 0.036) domains (Figure 3)14.\n\nSeveral studies reporting QoL data stratified by CKD stage were identified, in addition to studies that examined the impact of demographic factors and comorbidities on the relationship between disease severity and QoL.\n\nOverall, later stages of CKD were associated with worse QoL, but the trends for physical QoL were slightly different from those for mental QoL. In a study by Roumelioti et al., patients receiving maintenance dialysis had a near-significant decrease (17.7%; P = 0.05) in the SF-36 physical functioning domain score compared with patients with advanced CKD (eGFR < 30 mL/min/1.73 m2)15. Similarly, in two US studies by McClellan et al. and Porter et al., decreasing eGFR was associated with lower SF-12 (P = 0.001) and SF-36 (P = 0.004) PCS scores, but there was no apparent relationship between eGFR and MCS scores (data shown in Extended data: Supplementary content 216–18. McClellan et al. reported results from an unadjusted analysis as well as from an analysis adjusted for sociodemographic status and comorbidities; a correlation between eGFR and SF-12 PCS score was identified in both analyses (P < 0.001)16. Porter et al. reported results from an unadjusted analysis only17.\n\nIn several studies, the presence of comorbidities affected whether worsening CKD severity was linked to reductions in QoL. Porter et al. found that patients with a greater number of comorbidities experienced overall worse physical and mental QoL, assessed by SF-36 scores, than those with fewer comorbidities (mean PCS/MCS score: no comorbidities, 43.0/47.9; one comorbidity, 39.3/45.8; two or three comorbidities, 36.8/44.4; four or more comorbidities, 31.2/36.0; P < 0.001 for PCS and MCS)17. Furthermore, studies conducted by Campbell et al. and Wolfgram et al. in the USA found that adjustment for comorbidities and sociodemographic factors affected the relationship between QoL scores and eGFR19,20. Campbell et al. found that in an unadjusted analysis, patients with an eGFR of less than 60 mL/min/1.73 m2 reported a statistically significant reduction in SF-8 PCS scores (P < 0.05) compared with patients with an eGFR greater than 90 mL/min/1.72 m2 (reference group), whereas those with an eGFR of 60–89 mL/min/1.72 m2 reported higher SF-8 PCS scores (P < 0.05) than the reference group [mean PCS score by eGFR (mL/min/1.72 m2): ≥ 90, 44.04; 75–89, 44.97; 60–74, 45.00; 45–59, 42.32; 30–44, 40.41; ≤ 29, 40.02]19. However, following full adjustment for demographic factors and comorbidities, there were no significant differences between patients in any eGFR category and the reference group (Extended data: Supplementary content 2)18. Interestingly, there was a statistically significant difference in SF-8 MCS scores between patients who had the most severe disease (eGFR less than 29 mL/min/1.73 m2) and patients with the least severe disease in both unadjusted and adjusted analyses (fully adjusted analysis data shown in (Extended data: Supplementary content 2)18,19. Similarly, a study by Wolfgram et al., also in the USA, found that decreasing eGFR was associated with significantly lower EQ-5D scores in both the unadjusted analysis [mean EQ-5D score by eGFR (mL/min/1.72 m2): ≥ 60, 0.85; > 44 to < 60, 0.85; ≥ 44, 0.82] and an analysis adjusted for sociodemographic factors [mean EQ-5D score by eGFR (mL/min/1.72 m2): ≥ 60, 0.84; > 44 to < 60, 0.84; ≥ 44, 0.82]20. However, in an analysis adjusted for sociodemographics and comorbidities, including CVD, no relationship between eGFR and QoL was found [mean EQ-5D score by eGFR (mL/min/1.72 m2): ≥ 60, 0.73; > 44 to < 60, 0.75; ≥ 44, 0.73]20.\n\nStudies by Rajan et al. and Eriksson et al. assessed the impact of increasing disease severity in patient populations with specific comorbidities21,22. Rajan et al. reported QoL scores for patients with DM that was either prevalent (longer than 3 years) or recent-onset (less than 3 years)22. In both patient populations, scores for the physical functioning and role-limiting (physical) domains of the SF-36 were lower with increasing CKD stage; however, scores for the mental health domain increased up to stage 3, but were lower for stage 4 and lower still for stage 5 (Extended data: Supplementary content 2)18,22. The authors acknowledged that many of the patients with CKD stage 0/1 had claims for International Classification of Diseases (Ninth Revision) codes relating to mental illness, which may account for the lower scores in the mental health domain in this subgroup22. Eriksson et al. reported QoL in patients with pre-dialysis CKD (stage 3–4) or receiving dialysis, with or without anaemia21. Lower SF-12 and EQ-5D scores were reported by patients with CKD stage 4 than those with CKD stage 3, and the lowest scores were reported by patients receiving dialysis; however, no statistical analysis of the difference between stages was reported. This trend was present both in patients with anaemia and in those without anaemia (Figure 4)21.\n\nNo statistical analysis of the between-stage difference was reported. EQ-5D, 5-dimension EuroQol questionnaire. MCS, mental component summary. PCS, physical component summary. SF-12, 12-item Short-Form Health Survey.\n\nStudies were identified that reported QoL data associated with treatment modality and setting8,12,23–41; comorbidities in CKD17,20–22,41–60; mental health61,62; sleep quality10,59,63–65; patient support, care, and disease management11,66–83; sociodemographic factors84,85; and other factors predictive of CKD development15,86–103. These studies are listed in Extended data: Supplementary content 3104. A total of eight studies were identified on symptom burden in patients with CKD, of which two studies105,106 discussed the symptoms experienced by patients with CKD stage 5 who were not receiving dialysis, and six studies24,34,67,107–109 discussed symptom burden in patients receiving dialysis (Extended data: Supplementary content 3104).\n\nThe SR identified several studies that compared costs and resource use between individuals with CKD and those with normal kidney function. Some of the studies differentiated between patients with pre-dialysis CKD and those who required RRT, and the patients in several of the studies had additional comorbidities.\n\nAcross all studies, pre-dialysis CKD and ESRD requiring RRT were associated with significant increases in cost and resource use. Kim et al. found that patients with pre-dialysis CKD were significantly more likely to experience hip fracture-related hospitalization than patients with normal kidney function, and those receiving RRT were at higher risk than patients with pre-dialysis CKD. Median hospital costs were significantly greater in patients with CKD than for the general population and were highest for patients requiring RRT. This trend was also observed in the length of hospital stay, although the absolute differences between groups were small (Figure 5)110. Kumar et al. found very similar results in a study examining hospitalization rates and length of stay for pulmonary embolism in patient populations with normal kidney function, with pre-dialysis CKD or receiving dialysis (Figure 5)112. In a study of patients with hepatitis C, total healthcare costs were 2.9 times higher for those who had CKD, compared with those who had hepatitis C without CKD (USD 548 vs USD 1922; P < 0.001)112. Some patients with CKD in this study were defined as having ESRD; however, patients’ dialysis or transplant status was not specified. In a fourth study, patients with bone metastases and impaired kidney function incurred 60% higher total healthcare costs than a control group of patients with bone metastases and normal kidney function (USD 142,267 vs USD 88,839; P < 0.001). These increased costs were driven by hospital admission, emergency department and outpatient visits, longer length of stay in hospital, and higher pharmacy costs113. An Australian study by Wyld et al. also showed that patients with any stage of pre-dialysis CKD incurred significantly higher costs than individuals with no CKD (Figure 6a)114.\n\naP < 0.001 for each pairwise comparison. bIncluded patients receiving dialysis or renal transplant. cIncluded patients receiving dialysis. dP < 0.05 versus normal kidney function. CKD, chronic kidney disease. ESRD, end-stage renal disease. IQR, interquartile range. LoS, length of stay. USD, US dollars.\n\naOne-way ANOVA. bDialysis status not specified. cHypertension, DM, history of CHF, hyperkalaemia, or proteinuria. ANOVA, analysis of variance. AUD, Australian dollars. CHF, congestive heart failure. CKD, chronic kidney disease. DM, diabetes mellitus. EUR, euros. USD, US dollars.\n\nIn total, six studies reported costs for patient populations stratified by CKD stage. Across the evidence base, later-stage disease was associated with comparatively higher costs, both in studies including only patients with pre-dialysis CKD and in those in which some patients were receiving RRT.\n\nWyld et al. showed that costs increased significantly across pre-dialysis CKD stages (Figure 6a)114. Patients with CKD stage 3 incurred approximately 28% higher direct annual healthcare costs than those with CKD stage 1–2; however, there was a much larger cost increase for CKD stage 4–5, with patients in this group incurring more than fourfold higher costs than patients with CKD stage 3. This cost increase was apparent in subgroups of patients both with and without DM, suggesting that the high costs associated with CKD stage 4 and 5 were not attributable solely to the presence of DM. However, only 18 patients in this study had CKD stage 4–5, meaning that the results may have been skewed by a small number of individuals who incurred exceptionally high healthcare expenditure.\n\nTwo studies examined the costs of CKD across stages 4–5 or 3–5. An Italian study by Turchetti et al. of patients with pre-dialysis CKD showed a 31% increase in the direct annual medical costs associated with CKD stage 5, compared with CKD stage 4 (P < 0.01; Figure 6b)115. DM and CVD were shown to have an impact on costs incurred by patients at either CKD stage. Smith et al., who conducted a study in the USA, found that patients with CKD stage 3a who had comorbidities and patients with CKD stage 3b incurred similar monthly costs associated with health insurance claims (Figure 6c)116. Those with CKD stage 4–5, however, incurred more than double these costs (P < 0.05). Patients’ dialysis status was not specified in the study publication, but a breakdown of costs was reported. Later-stage CKD was associated with higher inpatient, outpatient, and professional services costs than CKD stage 3a or 3b, whereas the highest prescription costs were incurred by patients with CKD stage 3b.\n\nSeveral studies examining costs by CKD stage included patients who had progressed to RRT and therefore incurred additional costs117,118. Two studies were conducted in patient populations with CKD and DM in the USA. McQueen et al. found an increase in annual costs for each successive CKD stage, with the exception of CKD stage 2, which was associated with slightly lower costs than CKD stage 1. As in other studies, the largest increase was between the later disease stages, with a 71% increase in costs for stage 5 compared with stage 4 (Figure 7a)117. A similar cost increase was reported by Vupputuri et al. who compared costs between patients whose disease progressed and those who remained stable. Individuals who progressed to RRT incurred 77% higher annual medical costs than patients who remained at CKD stage 4 (Figure 7b)118. In a third study, Ariyaratne et al. examined the impact of CKD stage on patients undergoing percutaneous coronary intervention (PCI) in Australia. Direct cardiovascular costs, assessed 1 year after PCI, did not increase significantly for CKD stage 3 from stage 1–2 (AUD 4851 vs AUD 4442; 9% increase; P = 0.052), whereas patients with CKD stage 4–5, some of whom were receiving dialysis, incurred significantly higher costs than those at stage 1–2 (AUD 6958; 57% increase; P < 0.001)119.\n\na14.4% of patients at CKD stage 4 had a procedure code for dialysis during the baseline period. b70.3% of patients at CKD stage 5 had a procedure code for dialysis during the baseline period. cESRD was defined as a requirement for dialysis or renal transplant, and was considered to be equivalent to CKD stage 5 in this study. CKD, chronic kidney disease. ESRD, end-stage renal disease. NS, nonsignificant. T2DM, type 2 diabetes mellitus. USD, US dollars.\n\nTwo studies were identified that compared costs for patients with pre-dialysis CKD with those for patients receiving dialysis (Extended data: Supplementary content 4)120–122. Eriksson et al. compared costs and resource use in Sweden between four treatment groups: patients with pre-dialysis CKD (stage 4–5), those receiving haemodialysis, those receiving peritoneal dialysis, and patients with renal transplant. Patients with pre-dialysis CKD incurred the lowest total costs and had the lowest rate of outpatient visits of any treatment group, but had a slightly higher hospitalization rate and a greater mean number of hospital days annually than those who had received a renal transplant121. Both types of dialysis incurred higher inpatient and outpatient costs than pre-dialysis CKD or renal transplant, with more than 70% of the costs associated with haemodialysis contributed by outpatient care. All types of RRT were associated with additional expenditure on drugs, compared with pre-dialysis CKD. In addition, a study by Escudero-Vilaplana et al. showed that among patients receiving erythropoietin-stimulating agents (ESAs), the monthly cost of ESA therapy was significantly higher for patients receiving peritoneal dialysis than for those with pre-dialysis CKD (stage 2–5)122.\n\nThe other studies identified in the economic burden SR did not compare patients with CKD with the general population or report data by disease severity. Data from these studies are not reported in this manuscript; however, the studies are listed in Extended data: Supplementary content 3104 to illustrate key trends and groupings in the economic evidence base for CKD. The studies that we identified reported data on the costs associated with different treatment modalities or settings26,121,123–127; on the cost of comorbidities in CKD, including DM, cardiovascular complications, and anaemia112–115,117–119,122,128–141; on hospitalization costs and rates for patients with CKD62,66,142–148; and on other costs in CKD149–155.\n\n\nDiscussion\n\nThe studies identified in these SRs clearly illustrate the humanistic and economic impact of CKD. Patients with pre-dialysis CKD as well as patients who require RRT are likely to have worse QoL than the general population, and also incur higher healthcare costs. Several studies indicated that increasing CKD severity is associated with a gradual reduction in physical QoL; however, evidence for the impact of CKD on mental QoL was inconsistent. The economic burden SR identified strong evidence that costs and resource use are higher for patients with CKD than for the general population. Costs are especially high for patients at the most severe stage of CKD, for whom dialysis is often required. In patients with pre-dialysis CKD, costs increase incrementally with disease severity, particularly when comparing CKD stages 4–5 with stages 2–3. These findings underline the importance of early intervention in CKD to prevent patients from progressing to late-stage CKD and dialysis.\n\nSeveral studies identified in these SRs report data concerning the impact of comorbidities on QoL or costs. It is likely that some of the humanistic and economic burden associated with CKD, particularly later-stage disease, is linked to comorbidities. Patients with CKD may be more likely to have comorbidities than the general population; furthermore, CKD is itself a risk factor for several complications and comorbidities, which contribute to the effect that the disease has on long-term outcomes. By examining subgroups of patients with particular comorbidities, as in the study by Wyld et al., it is possible to gain an understanding of the relative contribution of various comorbidities to overall disease burden. However, in order to study the burden of CKD specifically, analyses should be adjusted only for comorbidities that are unrelated to or are risk factors for CKD, but not comorbidities that are usually consequences of CKD. Exact differentiation between these types of comorbidities is not always possible, which can confound attempts to determine the true burden of CKD as distinct from co-existing conditions.\n\nIn this review, the impact of disease severity has been inferred almost exclusively from cross-sectional data; only one longitudinal study was identified. The economic study by Vupputuri et al. examined the impact and long-term consequences of increasing CKD severity in a patient cohort over time by comparing patients who remained at the same CKD stage and those whose disease progressed; however, the remaining economic studies and all QoL studies were cross-sectional. A longitudinal cohort study of patients with early-stage CKD could provide additional insights into the within-patient effects of CKD progression, the development of comorbidities and complications, and the competing risk of death, helping to quantify the extent to which this results in over-representation of relatively young patients or patients with few comorbidities in the later CKD stages, due to their better overall survival.\n\nMoreover, definitions of CKD stage and grouping of patients into categories were not consistent across the evidence base, and in some cases eGFR category was used as a measure for disease severity but did not correspond exactly to the CKD stages used in other studies. Patients’ dialysis status differed between studies, as did the definition of ESRD, which was defined as the requirement for dialysis only in some studies and was expanded to include renal transplant in others. Similarly, CKD stage 5 was considered a pre-dialysis disease stage in some studies, but in others was analogous to RRT. For QoL, the use of different instruments across studies also contributed to the difficulty in drawing comparisons between different data sets. Therefore, it was not possible to strengthen our findings by performing a meta-analysis using the data identified.\n\nThese SRs identified a broad range of studies reporting QoL or cost and resource use data for patients with CKD, including the impact of treatment modality and the effect of comorbidities. In the majority of studies, however, the scope of the data reported was relatively limited or comparisons were made between two groups of patients with CKD, differentiated by other factors, such as sociodemographic characteristics or treatment; these studies were outside the scope of this review. Owing to the design of the SRs, in which restrictions were applied to publication type, population size, and QoL instrument, it is also possible that some studies of interest may not have been examined in detail. Furthermore, no assessment of risk of bias was performed.\n\n\nConclusions\n\nOur findings indicate that the development and progression of CKD are associated with both a reduction in patients’ QoL and an increase in healthcare costs. Interventions and initiatives to prevent CKD progression, especially to later stages of disease and the requirement for dialysis, could improve patients’ well-being and may limit the growing economic burden of CKD. We have also identified the need for further research, particularly longitudinal studies, as well as studies that collect full information on patients’ disease history, treatment status, and comorbidities, and adjust for these factors when necessary. Such studies will be vital to quantify the impact of slowing CKD progression on the disease’s humanistic and economic burden.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: Freeman et al. Supplementary content 1 – Table S1. http://doi.org/10.6084/m9.figshare.100114766.\n\nThis project contains Table S1: Electronic search strategy.\n\nFigshare: Freeman et al. Supplementary content 2 – Figure S1. https://doi.org/10.6084/m9.figshare.1001168318.\n\nThis project contains Figure S1: Physical and mental component summary scores with increasing severity of chronic kidney disease.\n\nFigshare: Freeman et al. Supplementary content 3 – Table S2. http://doi.org/10.6084/m9.figshare.10011692104.\n\nThis project contains Table S2: Additional quality of life, symptom burden and cost and resource use studies identified in the systematic review.\n\nFigshare: Freeman et al. Supplementary content 4 – Table S3. https://doi.org/10.6084/m9.figshare.10011710120.\n\nThis project contains Table S3: Summary of studies that compared costs for patients with chronic kidney disease by renal replacement therapy status.\n\nFigshare: Freeman et al. Supplementary content 5 – Protocol. https://doi.org/10.6084/m9.figshare.11112578156.\n\nThis project contains the protocol for this systematic review.\n\nFigshare: PRISMA checklist for ‘Humanistic burden and economic impact of chronic kidney disease: a systematic literature review’. http://doi.org/10.6084/m9.figshare.10011893157.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nHill NR, Fatoba ST, Oke JL, et al.: Global Prevalence of Chronic Kidney Disease - A Systematic Review and Meta-Analysis. PLoS One. 2016; 11(7): e0158765. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKazancioğlu R: Risk factors for chronic kidney disease: an update. Kidney Int Suppl (2011). 2013; 3(4): 368–71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUnited States Renal Data System: Chapter 1: CKD in the general population. (Accessed 9 November 2017). 2016. Reference Source\n\nKhan S, Amedia CA Jr: Economic burden of chronic kidney disease. J Eval Clin Pract. 2008; 14(3): 422–34. PubMed Abstract \| Publisher Full Text\n\nPoppe C, Crombez G, Hanoulle I, et al.: Improving quality of life in patients with chronic kidney disease: influence of acceptance and personality. Nephrol Dial Transplant. 2013; 28(1): 116–21. PubMed Abstract \| Publisher Full Text\n\nFreeman C, Giles L, Field P, et al.: Supplementary content 1 – Table S1. figshare. http://www.doi.org/10.6084/m9.figshare.10011476\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. BMJ. 2009; 339: b2535. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDavison SN, Jhangri GS, Feeny DH: Comparing the Health Utilities Index Mark 3 (HUI3) with the Short Form-36 preference-based SF-6D in chronic kidney disease. Value Health. 2009; 12(2): 340–5. PubMed Abstract \| Publisher Full Text\n\nNguyen HA, Anderson CAM, Miracle CM, et al.: The Association between Depression, Perceived Health Status, and Quality of Life among Individuals with Chronic Kidney Disease: An Analysis of the National Health and Nutrition Examination Survey 2011-2012. Nephron. 2017; 136(2): 127–35. PubMed Abstract \| Publisher Full Text\n\nAgarwal R, Light RP: Sleep and activity in chronic kidney disease: a longitudinal study. Clin J Am Soc Nephrol. 2011; 6(6): 1258–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoini S, Frimat L, Kessler M, et al.: Predialysis therapeutic care and health-related quality of life at dialysis onset (The pharmacoepidemiologic AVENIR study). Health Qual Life Outcomes. 2011; 9: 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWan EY, Chen JY, Choi EP, et al.: Patterns of health-related quality of life and associated factors in Chinese patients undergoing haemodialysis. Health Qual Life Outcomes. 2015; 13: 108. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang R, Tang C, Chen X, et al.: Poor sleep and reduced quality of life were associated with symptom distress in patients receiving maintenance hemodialysis. Health Qual Life Outcomes. 2016; 14(1): 125. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYong DS, Kwok AO, Wong DM, et al.: Symptom burden and quality of life in end-stage renal disease: a study of 179 patients on dialysis and palliative care. Palliat Med. 2009; 23(2): 111–19. PubMed Abstract \| Publisher Full Text\n\nRoumelioti ME, Argyropoulos C, Buysse DJ, et al.: Sleep quality, mood, alertness and their variability in CKD and ESRD. Nephron Clin Pract. 2010; 114(4): c277–87. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcClellan WM, Abramson J, Newsome B, et al.: Physical and psychological burden of chronic kidney disease among older adults. Am J Nephrol. 2010; 31(4): 309–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPorter A, Fischer MJ, Brooks D, et al.: Quality of life and psychosocial factors in African Americans with hypertensive chronic kidney disease. Transl Res. 2012; 159(1): 4–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFreeman C, Giles L, Field P, et al.: Supplementary content 2 – Figure S1. figshare. http://www.doi.org/10.6084/m9.figshare.10011683\n\nCampbell KH, Huang ES, Dale W, et al.: Association between estimated GFR, health-related quality of life, and depression among older adults with diabetes: the Diabetes and Aging Study. Am J Kidney Dis. 2013; 62(3): 541–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWolfgram DF, Garcia K, Evans G, et al.: Association of Albuminuria and Estimated Glomerular Filtration Rate with Functional Performance Measures in Older Adults with Chronic Kidney Disease. Am J Nephrol. 2017; 45(2): 172–9. PubMed Abstract \| Publisher Full Text\n\nEriksson D, Goldsmith D, Teitsson S, et al.: Cross-sectional survey in CKD patients across Europe describing the association between quality of life and anaemia. BMC Nephrol. 2016; 17(1): 97. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRajan M, Lai KC, Tseng CL, et al.: Estimating utilities for chronic kidney disease, using SF-36 and SF-12-based measures: challenges in a population of veterans with diabetes. Qual Life Res. 2013; 22(1): 53–64. PubMed Abstract \| Publisher Full Text\n\nBalasubramanian G, McKitty K, Fan SL: Comparing automated peritoneal dialysis with continuous ambulatory peritoneal dialysis: survival and quality of life differences? Nephrol Dial Transplant. 2011; 26(5): 1702–8. PubMed Abstract \| Publisher Full Text\n\nBrown EA, Johansson L, Farrington K, et al.: Broadening Options for Long-term Dialysis in the Elderly (BOLDE): differences in quality of life on peritoneal dialysis compared to haemodialysis for older patients. Nephrol Dial Transplant. 2010; 25(11): 3755–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen JY, Wan EY, Choi EP, et al.: Clinical and patient-reported outcomes of Chinese patients undergoing haemodialysis in hospital or in the community: A 1-year longitudinal study. Nephrology (Carlton). 2016; 21(7): 617–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCortés-Sanabria L, Paredes-Ceseña CA, Herrera-Llamas RM, et al.: Comparison of cost-utility between automated peritoneal dialysis and continuous ambulatory peritoneal dialysis. Arch Med Res. 2013; 44(8): 655–61. PubMed Abstract \| Publisher Full Text\n\nDavison SN, Jhangri GS: Existential and religious dimensions of spirituality and their relationship with health-related quality of life in chronic kidney disease. Clin J Am Soc Nephrol. 2010; 5(11): 1969–76. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDavison SN, Jhangri GS: The relationship between spirituality, psychosocial adjustment to illness, and health-related quality of life in patients with advanced chronic kidney disease. J Pain Symptom Manage. 2013; 45(2): 170–8. PubMed Abstract \| Publisher Full Text\n\nDiamant MJ, Harwood L, Movva S, et al.: A comparison of quality of life and travel-related factors between in-center and satellite-based hemodialysis patients. Clin J Am Soc Nephrol. 2010; 5(2): 268–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFinkelstein FO, Schiller B, Daoui R, et al.: At-home short daily hemodialysis improves the long-term health-related quality of life. Kidney Int. 2012; 82(5): 561–9. PubMed Abstract \| Publisher Full Text\n\nGarg AX, Suri RS, Eggers P, et al.: Patients receiving frequent hemodialysis have better health-related quality of life compared to patients receiving conventional hemodialysis. Kidney Int. 2017; 91(3): 746–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHill KE, Kim S, Crail S, et al.: A comparison of self-reported quality of life for an Australian haemodialysis and haemodiafiltration cohort. Nephrology (Carlton). 2017; 22(8): 624–30. PubMed Abstract \| Publisher Full Text\n\nIyasere OU, Brown EA, Johansson L, et al.: Quality of Life and Physical Function in Older Patients on Dialysis: A Comparison of Assisted Peritoneal Dialysis with Hemodialysis. Clin J Am Soc Nephrol. 2016; 11(3): 423–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLowney AC, Myles HT, Bristowe K, et al.: Understanding What Influences the Health-Related Quality of Life of Hemodialysis Patients: A Collaborative Study in England and Ireland. J Pain Symptom Manage. 2015; 50(6): 778–85. PubMed Abstract \| Publisher Full Text\n\nMazairac AH, de Wit GA, Grooteman MP, et al.: Effect of hemodiafiltration on quality of life over time. Clin J Am Soc Nephrol. 2013; 8(1): 82–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMichels WM, van Dijk S, Verduijn M, et al.: Quality of life in automated and continuous ambulatory peritoneal dialysis. Perit Dial Int. 2011; 31(2): 138–47. PubMed Abstract \| Publisher Full Text\n\nMoist LM, Bragg-Gresham JL, Pisoni RL, et al.: Travel time to dialysis as a predictor of health-related quality of life, adherence, and mortality: the Dialysis Outcomes and Practice Patterns Study (DOPPS). Am J Kidney Dis. 2008; 51(4): 641–50. PubMed Abstract \| Publisher Full Text\n\nMorena M, Jaussent A, Chalabi L, et al.: Treatment tolerance and patient-reported outcomes favor online hemodiafiltration compared to high-flux hemodialysis in the elderly. Kidney Int. 2017; 91(6): 1495–509. PubMed Abstract \| Publisher Full Text\n\nRayner HC, Zepel L, Fuller DS, et al.: Recovery time, quality of life, and mortality in hemodialysis patients: the Dialysis Outcomes and Practice Patterns Study (DOPPS). Am J Kidney Dis. 2014; 64(1): 86–94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaad MM, El Douaihy Y, Boumitri C, et al.: Predictors of quality of life in patients with end-stage renal disease on hemodialysis. Int J Nephrol Renovasc Dis. 2015; 8: 119–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTessari G, Dalle Vedove C, Loschiavo C, et al.: The impact of pruritus on the quality of life of patients undergoing dialysis: a single centre cohort study. J Nephrol. 2009; 22(2): 241–8. PubMed Abstract\n\nAbreo AP, Herzog CA, Kutner NG, et al.: Estimated pulmonary artery systolic pressure and self-reported physical function in patients on hemodialysis. Am J Nephrol. 2015; 41(4–5): 313–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAgarwal R, Kariyanna SS, Light RP: Circadian blood pressure classification scheme and the health of patients with chronic kidney disease. Am J Nephrol. 2009; 30(6): 536–46. PubMed Abstract \| Publisher Full Text\n\nAnand S, Kaysen GA, Chertow GM, et al.: Vitamin D deficiency, self-reported physical activity and health-related quality of life: the Comprehensive Dialysis Study. Nephrol Dial Transplant. 2011; 26(11): 3683–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlair D, Byham-Gray L, Lewis E, et al.: Prevalence of vitamin D [25(OH)D] deficiency and effects of supplementation with ergocalciferol (vitamin D2) in stage 5 chronic kidney disease patients. J Ren Nutr. 2008; 18(4): 375–82. PubMed Abstract \| Publisher Full Text\n\nBross R, Zitterkoph J, Pithia J, et al.: Association of serum total iron-binding capacity and its changes over time with nutritional and clinical outcomes in hemodialysis patients. Am J Nephrol. 2009; 29(6): 571–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFischer MJ, Kimmel PL, Greene T, et al.: Sociodemographic factors contribute to the depressive affect among African Americans with chronic kidney disease. Kidney Int. 2010; 77(11): 1010–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFoley RN, Curtis BM, Parfrey PS: Erythropoietin therapy, hemoglobin targets, and quality of life in healthy hemodialysis patients: a randomized trial. Clin J Am Soc Nephrol. 2009; 4(4): 726–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohansen KL, Dalrymple LS, Delgado C, et al.: Comparison of self-report-based and physical performance-based frailty definitions among patients receiving maintenance hemodialysis. Am J Kidney Dis. 2014; 64(4): 600–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrishnasamy R, Hawley CM, Stanton T, et al.: Association between left ventricular global longitudinal strain, health-related quality of life and functional capacity in chronic kidney disease patients with preserved ejection fraction. Nephrology (Carlton). 2016; 21(2): 108–15. PubMed Abstract \| Publisher Full Text\n\nLi J, Guo Q, Lin J, et al.: Prevalence and Associated Factors of Uraemic Pruritus in Continuous Ambulatory Peritoneal Dialysis Patients. Intern Med. 2015; 54(22): 2827–33. PubMed Abstract \| Publisher Full Text\n\nLiu T, Liang KV, Rosenbaum A, et al.: Peripheral vascular disease severity impacts health outcomes and health-related quality of life in maintenance hemodialysis patients in the HEMO Study. Nephrol Dial Transplant. 2012; 27(7): 2929–36. PubMed Abstract \| Publisher Full Text\n\nOsthus TB, von der Lippe N, Ribu L, et al.: Health-related quality of life and all-cause mortality in patients with diabetes on dialysis. BMC Nephrol. 2012; 13: 78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPlantinga LC, Fink NE, Jaar BG, et al.: Relation between level or change of hemoglobin and generic and disease-specific quality of life measures in hemodialysis. Qual Life Res. 2007; 16(5): 755–65. PubMed Abstract \| Publisher Full Text\n\nRamakrishnan K, Bond TC, Claxton A, et al.: Clinical characteristics and outcomes of end-stage renal disease patients with self-reported pruritus symptoms. Int J Nephrol Renovasc Dis. 2013; 7: 1–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRitz E, Laville M, Bilous RW, et al.: Target level for hemoglobin correction in patients with diabetes and CKD: primary results of the Anemia Correction in Diabetes (ACORD) Study. Am J Kidney Dis. 2007; 49(2): 194–207. PubMed Abstract \| Publisher Full Text\n\nSorensen EP, Sarnak MJ, Tighiouart H, et al.: The kidney disease quality of life cognitive function subscale and cognitive performance in maintenance hemodialysis patients. Am J Kidney Dis. 2012; 60(3): 417–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang X, Shen B, Zhuang X, et al.: Investigating Factors Associated with Depressive Symptoms of Chronic Kidney Diseases in China with Type 2 Diabetes. J Diabetes Res. 2017; 2017: 1769897. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeiss M, Mettang T, Tschulena U, et al.: Health-related quality of life in haemodialysis patients suffering from chronic itch: results from GEHIS (German Epidemiology Haemodialysis Itch Study). Qual Life Res. 2016; 25(12): 3097–106. PubMed Abstract \| Publisher Full Text\n\nWikström B: Itchy skin--a clinical problem for haemodialysis patients. Nephrol Dial Transplant. 2007; 22(Suppl 5): v3–7. PubMed Abstract \| Publisher Full Text\n\nBelayev LY, Mor MK, Sevick MA, et al.: Longitudinal associations of depressive symptoms and pain with quality of life in patients receiving chronic hemodialysis. Hemodial Int. 2015; 19(2): 216–24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLacson E Jr, Bruce L, Li NC, et al.: Depressive affect and hospitalization risk in incident hemodialysis patients. Clin J Am Soc Nephrol. 2014; 9(10): 1713–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrekke FB, Amro A, Hortemo Østhus TB, et al.: Sleep complaints, depression and quality of life in Norwegian dialysis patients. Clin Nephrol. 2013; 80(2): 88–97. PubMed Abstract \| Publisher Full Text\n\nKumar B, Tilea A, Gillespie BW, et al.: Significance of self-reported sleep quality (SQ) in chronic kidney disease (CKD): the Renal Research Institute (RRI)-CKD study. Clin Nephrol. 2010; 73(2): 104–14. PubMed Abstract \| Publisher Full Text\n\nKutner NG, Zhang R, Huang Y, et al.: Association of sleep difficulty with Kidney Disease Quality of Life cognitive function score reported by patients who recently started dialysis. Clin J Am Soc Nephrol. 2007; 2(2): 284–9. PubMed Abstract \| Publisher Full Text\n\nBennett PN, Fraser S, Barnard R, et al.: Effects of an intradialytic resistance training programme on physical function: a prospective stepped-wedge randomized controlled trial. Nephrol Dial Transplant. 2016; 31(8): 1302–9. PubMed Abstract \| Publisher Full Text\n\nBerman N, Christianer K, Roberts J, et al.: Disparities in symptom burden and renal transplant eligibility: a pilot study. J Palliat Med. 2013; 16(11): 1459–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlakeman T, Blickem C, Kennedy A, et al.: Effect of information and telephone-guided access to community support for people with chronic kidney disease: randomised controlled trial. PLoS One. 2014; 9(10): e109135. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrown MA, Collett GK, Josland EA, et al.: CKD in elderly patients managed without dialysis: survival, symptoms, and quality of life. Clin J Am Soc Nephrol. 2015; 10(2): 260–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFeldt-Rasmussen B, Lange M, Sulowicz W, et al.: Growth hormone treatment during hemodialysis in a randomized trial improves nutrition, quality of life, and cardiovascular risk. J Am Soc Nephrol. 2007; 18(7): 2161–71. PubMed Abstract \| Publisher Full Text\n\nGriva K, Jayasena D, Davenport A, et al.: Illness and treatment cognitions and health related quality of life in end stage renal disease. Br J Health Psychol. 2009; 14(Pt 1): 17–34. PubMed Abstract \| Publisher Full Text\n\nLewis EF, Pfeffer MA, Feng A, et al.: Darbepoetin alfa impact on health status in diabetes patients with kidney disease: a randomized trial. Clin J Am Soc Nephrol. 2011; 6(4): 845–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi B, Cairns JA, Draper H, et al.: Estimating Health-State Utility Values in Kidney Transplant Recipients and Waiting-List Patients Using the EQ-5D-5L. Value Health. 2017; 20(7): 976–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi J, Wang H, Xie H, et al.: Effects of post-discharge nurse-led telephone supportive care for patients with chronic kidney disease undergoing peritoneal dialysis in China: a randomized controlled trial. Perit Dial Int. 2014; 34(3): 278–88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeuleman Y, Hoekstra T, Dekker FW, et al.: Sodium Restriction in Patients With CKD: A Randomized Controlled Trial of Self-management Support. Am J Kidney Dis. 2017; 69(5): 576–86. PubMed Abstract \| Publisher Full Text\n\nPerl J, Zhang J, Gillespie B, et al.: Reduced survival and quality of life following return to dialysis after transplant failure: the Dialysis Outcomes and Practice Patterns Study. Nephrol Dial Transplant. 2012; 27(12): 4464–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPlantinga LC, Fink NE, Harrington-Levey R, et al.: Association of social support with outcomes in incident dialysis patients. Clin J Am Soc Nephrol. 2010; 5(8): 1480–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRodrigue JR, Mandelbrot DA, Hanto DW, et al.: A cross-sectional study of fatigue and sleep quality before and after kidney transplantation. Clin Transplant. 2011; 25(1): E13–21. PubMed Abstract \| Publisher Full Text\n\nRossi AP, Burris DD, Lucas FL, et al.: Effects of a renal rehabilitation exercise program in patients with CKD: a randomized, controlled trial. Clin J Am Soc Nephrol. 2014; 9(12): 2052–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSuri RS, Larive B, Garg AX, et al.: Burden on caregivers as perceived by hemodialysis patients in the Frequent Hemodialysis Network (FHN) trials. Nephrol Dial Transplant. 2011; 26(7): 2316–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThilly N, Boini S, Soudant M, et al.: Outcomes of patients with delayed dialysis initiation: Results from the AVENIR study. Am J Nephrol. 2011; 33(1): 76–83. PubMed Abstract \| Publisher Full Text\n\nvon der Lippe N, Waldum B, Brekke FB, et al.: From dialysis to transplantation: a 5-year longitudinal study on self-reported quality of life. BMC Nephrol. 2014; 15: 191. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvon der Lippe N, Waldum B, Østhus TB, et al.: Health related quality of life in patients in dialysis after renal graft loss and effect of gender. BMC Womens Health. 2014; 14(1): 34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGemmell LA, Terhorst L, Jhamb M, et al.: Gender and Racial Differences in Stress, Coping, and Health-Related Quality of Life in Chronic Kidney Disease. J Pain Symptom Manage. 2016; 52(6): 806–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRicardo AC, Hacker E, Lora CM, et al.: Validation of the Kidney Disease Quality of Life Short Form 36 (KDQOL-36) US Spanish and English versions in a cohort of Hispanics with chronic kidney disease. Ethn Dis. 2013; 23(2): 202–9. PubMed Abstract \| Free Full Text\n\nAmro A, Waldum B, von der Lippe N, et al.: Symptom clusters predict mortality among dialysis patients in Norway: a prospective observational cohort study. J Pain Symptom Manage. 2015; 49(1): 27–35. PubMed Abstract \| Publisher Full Text\n\nChan MF, Wong FK, Chow SK: Investigating the health profile of patients with end-stage renal failure receiving peritoneal dialysis: a cluster analysis. J Clin Nurs. 2010; 19(5–6): 649–57. PubMed Abstract \| Publisher Full Text\n\nEnia G, Torino C, Panuccio V, et al.: Asymptomatic pulmonary congestion and physical functioning in hemodialysis patients. Clin J Am Soc Nephrol. 2013; 8(8): 1343–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFeroze U, Noori N, Kovesdy CP, et al.: Quality-of-life and mortality in hemodialysis patients: roles of race and nutritional status. Clin J Am Soc Nephrol. 2011; 6(5): 1100–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHayashino Y, Fukuhara S, Akiba T, et al.: Low health-related quality of life is associated with all-cause mortality in patients with diabetes on haemodialysis: the Japan Dialysis Outcomes and Practice Pattern Study. Diabet Med. 2009; 26(9): 921–7. PubMed Abstract \| Publisher Full Text\n\nJhamb M, Argyropoulos C, Steel JL, et al.: Correlates and outcomes of fatigue among incident dialysis patients. Clin J Am Soc Nephrol. 2009; 4(11): 1779–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJhamb M, Pike F, Ramer S, et al.: Impact of fatigue on outcomes in the hemodialysis (HEMO) study. Am J Nephrol. 2011; 33(6): 515–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKalantar-Zadeh K, Kopple JD, Kamranpour N, et al.: HDL-inflammatory index correlates with poor outcome in hemodialysis patients. Kidney Int. 2007; 72(9): 1149–56. PubMed Abstract \| Publisher Full Text\n\nLacson E Jr, Xu J, Lin SF, et al.: Association between achievement of hemodialysis quality-of-care indicators and quality-of-life scores. Am J Kidney Dis. 2009; 54(6): 1098–107. PubMed Abstract \| Publisher Full Text\n\nLaegreid IK, Aasarod K, Bye A, et al.: The impact of nutritional status, physical function, comorbidity and early versus late start in dialysis on quality of life in older dialysis patients. Ren Fail. 2014; 36(1): 9–16. PubMed Abstract \| Publisher Full Text\n\nLo JC, Beck GJ, Kaysen GA, et al.: Hyperprolactinemia in end-stage renal disease and effects of frequent hemodialysis. Hemodial Int. 2017; 21(2): 190–6. PubMed Abstract \| Publisher Full Text\n\nLoosman WL, Hoekstra T, van Dijk S, et al.: Short-Form 12 or Short-Form 36 to measure quality-of-life changes in dialysis patients? Nephrol Dial Transplant. 2015; 30(7): 1170–6. PubMed Abstract \| Publisher Full Text\n\nMazairac AH, de Wit GA, Penne EL, et al.: Protein-energy nutritional status and kidney disease-specific quality of life in hemodialysis patients. J Ren Nutr. 2011; 21(5): 376–86.e1. PubMed Abstract \| Publisher Full Text\n\nØsthus TB, Preljevic VT, Sandvik L, et al.: Mortality and health-related quality of life in prevalent dialysis patients: Comparison between 12-items and 36-items short-form health survey. Health Qual Life Outcomes. 2012; 10: 46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPorter A, Fischer MJ, Wang X, et al.: Quality of life and outcomes in African Americans with CKD. J Am Soc Nephrol. 2014; 25(8): 1849–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobinson-Cohen C, Hall YN, Katz R, et al.: Self-rated health and adverse events in CKD. Clin J Am Soc Nephrol. 2014; 9(12): 2044–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThomas CJ, Washington TA: Religiosity and social support: implications for the health-related quality of life of African American hemodialysis patients. J Relig Health. 2012; 51(4): 1375–85. PubMed Abstract \| Publisher Full Text\n\nUnruh ML, Newman AB, Larive B, et al.: The influence of age on changes in health-related quality of life over three years in a cohort undergoing hemodialysis. J Am Geriatr Soc. 2008; 56(9): 1608–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFreeman C, Giles L, Field P, et al.: Supplementary content 3 – Table S2. figshare. http://www.doi.org/10.6084/m9.figshare.10011692\n\nBrennan F, Collett G, Josland EA, et al.: The symptoms of patients with CKD stage 5 managed without dialysis. Prog Palliat Care. 2015; 23(5): 267–73. Publisher Full Text\n\nMurtagh FE, Addington-Hall JM, Edmonds PM, et al.: Symptoms in advanced renal disease: a cross-sectional survey of symptom prevalence in stage 5 chronic kidney disease managed without dialysis. J Palliat Med. 2007; 10(6): 1266–76. PubMed Abstract \| Publisher Full Text\n\nCano AE, Neil AK, Kang JY, et al.: Gastrointestinal symptoms in patients with end-stage renal disease undergoing treatment by hemodialysis or peritoneal dialysis. Am J Gastroenterol. 2007; 102(9): 1990–7. PubMed Abstract\n\nCaplin B, Kumar S, Davenport A: Patients' perspective of haemodialysis-associated symptoms. Nephrol Dial Transplant. 2011; 26(8): 2656–63. PubMed Abstract \| Publisher Full Text\n\nDong R, Guo ZY, Ding JR, et al.: Gastrointestinal symptoms: a comparison between patients undergoing peritoneal dialysis and hemodialysis. World J Gastroenterol. 2014; 20(32): 11370–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim SM, Long J, Montez-Rath M, et al.: Hip Fracture in Patients With Non-Dialysis-Requiring Chronic Kidney Disease. J Bone Miner Res. 2016; 31(10): 1803–9. PubMed Abstract \| Publisher Full Text\n\nKumar G, Sakhuja A, Taneja A, et al.: Pulmonary embolism in patients with CKD and ESRD. Clin J Am Soc Nephrol. 2012; 7(10): 1584–90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPuenpatom A, Hull M, McPheeters J, et al.: Disease Burden, Early Discontinuation, and Healthcare Costs in Hepatitis C Patients with and without Chronic Kidney Disease Treated with Interferon-Free Direct-Acting Antiviral Regimens. Clin Drug Investig. 2017; 37(7): 687–97. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQian Y, Arellano J, Bhowmik D, et al.: Healthcare resource use and costs associated with renal impairment in US patients with bone metastases from solid tumors. J Oncol Pharm Pract. 2017; 23(3): 195–202. PubMed Abstract \| Publisher Full Text\n\nWyld ML, Lee CM, Zhuo X, et al.: Cost to government and society of chronic kidney disease stage 1-5: a national cohort study. Intern Med J. 2015; 45(7): 741–7. PubMed Abstract \| Publisher Full Text\n\nTurchetti G, Bellelli S, Amato M, et al.: The social cost of chronic kidney disease in Italy. Eur J Health Econ. 2017; 18(7): 847–58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith ZG, McNicoll L, Clark TL, et al.: Medical Neighborhood Model for the Care of Chronic Kidney Disease Patients. Am J Nephrol. 2016; 44(4): 308–15. PubMed Abstract \| Publisher Full Text\n\nMcQueen RB, Farahbakhshian S, Bell KF, et al.: Economic burden of comorbid chronic kidney disease and diabetes. J Med Econ. 2017; 20(6): 585–91. PubMed Abstract \| Publisher Full Text\n\nVupputuri S, Kimes TM, Calloway MO, et al.: The economic burden of progressive chronic kidney disease among patients with type 2 diabetes. J Diabetes Complications. 2014; 28(1): 10–16. PubMed Abstract \| Publisher Full Text\n\nAriyaratne TV, Ademi Z, Duffy SJ, et al.: Cardiovascular readmissions and excess costs following percutaneous coronary intervention in patients with chronic kidney disease: data from a large multi-centre Australian registry. Int J Cardiol. 2013; 168(3): 2783–90. PubMed Abstract \| Publisher Full Text\n\nFreeman C, Giles L, Field P, et al.: Supplementary content 4 – Table S3. figshare. http://www.doi.org/10.6084/m9.figshare.10011710\n\nEriksson JK, Neovius M, Jacobson SH, et al.: Healthcare costs in chronic kidney disease and renal replacement therapy: a population-based cohort study in Sweden. BMJ Open. 2016; 6(10): e012062. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEscudero-Vilaplana V, Martínez-Nieto C, López-Gómez JM, et al.: Erythropoiesis-stimulating agents in anaemia due to chronic kidney disease: a cost-minimization analysis. Int J Clin Pharm. 2013; 35(3): 463–8. PubMed Abstract \| Publisher Full Text\n\nChow E, Wong H, Hahn-Goldberg S, et al.: Inpatient and emergent resource use of patients on dialysis at an academic medical center. Nephron Clin Pract. 2014; 126(3): 124–7. PubMed Abstract \| Publisher Full Text\n\nChui BK, Manns B, Pannu N, et al.: Health care costs of peritoneal dialysis technique failure and dialysis modality switching. Am J Kidney Dis. 2013; 61(1): 104–11. PubMed Abstract \| Publisher Full Text\n\nCortés-Sanabria L, Rodríguez-Arreola BE, Ortiz-Juárez VR, et al.: Comparison of direct medical costs between automated and continuous ambulatory peritoneal dialysis. Perit Dial Int. 2013; 33(6): 679–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCouillerot-Peyrondet AL, Sambuc C, Sainsaulieu Y, et al.: A comprehensive approach to assess the costs of renal replacement therapy for end-stage renal disease in France: the importance of age, diabetes status, and clinical events. Eur J Health Econ. 2017; 18(4): 459–69. PubMed Abstract \| Publisher Full Text\n\nSánchez-Escuredo A, Alsina A, Diekmann F, et al.: Economic analysis of the treatment of end-stage renal disease treatment: living-donor kidney transplantation versus hemodialysis. Transplant Proc. 2015; 47(1): 30–3. PubMed Abstract \| Publisher Full Text\n\nChen SY, Lee YC, Alas V, et al.: Outcomes associated with concordance of oral antidiabetic drug treatments to prescribing information in patients with type 2 diabetes mellitus and chronic kidney disease. J Med Econ. 2013; 16(5): 586–95. PubMed Abstract \| Publisher Full Text\n\nChiroli S, Mattin C, Belozeroff V, et al.: Impact of mineral and bone disorder on healthcare resource use and associated costs in the European Fresenius medical care dialysis population: a retrospective cohort study. BMC Nephrol. 2012; 13: 140. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHirth RA, Turenne MN, Wheeler JR, et al.: The initial impact of Medicare's new prospective payment system for kidney dialysis. Am J Kidney Dis. 2013; 62(4): 662–9. PubMed Abstract \| Publisher Full Text\n\nHörbrand F, Rottenkolber D, Fischaleck J, et al.: Erythropoietin-induced treatment costs in patients suffering from renal anemia - a comparison between biosimilar and originator drugs. Gesundheitswesen. 2014; 76(11): e79–84. PubMed Abstract \| Publisher Full Text\n\nJohnson DW, Pascoe EM, Badve SV, et al.: A randomized, placebo-controlled trial of pentoxifylline on erythropoiesis-stimulating agent hyporesponsiveness in anemic patients with CKD: the Handling Erythropoietin Resistance With Oxpentifylline (HERO) trial. Am J Kidney Dis. 2015; 65(1): 49–57. PubMed Abstract \| Publisher Full Text\n\nKadam UT, Uttley J, Jones PW, et al.: Chronic disease multimorbidity transitions across healthcare interfaces and associated costs: a clinical-linkage database study. BMJ Open. 2013; 3(7): pii: e003109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKarunaratne K, Stevens P, Irving J, et al.: The impact of pay for performance on the control of blood pressure in people with chronic kidney disease stage 3-5. Nephrol Dial Transplant. 2013; 28(8): 2107–16. PubMed Abstract \| Publisher Full Text\n\nKleophas W, Bieber B, Robinson BM, et al.: Implementation and first results of a German chronic kidney disease registry. Clin Nephrol. 2013; 79(3): 184–91. PubMed Abstract \| Publisher Full Text\n\nMannino DM, Higuchi K, Yu TC, et al.: Economic Burden of COPD in the Presence of Comorbidities. Chest. 2015; 148(1): 138–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMitri G, Wittbrodt ET, Turpin RS, et al.: Cost Comparison of Urate-Lowering Therapies in Patients with Gout and Moderate-to-Severe Chronic Kidney Disease. J Manag Care Spec Pharm. 2016; 22(4): 326–36. PubMed Abstract \| Publisher Full Text\n\nPockett RD, Cevro E, Chamberlain G, et al.: Assessment of resource use and costs associated with parathyroidectomy for secondary hyperparathyroidism in end stage renal disease in the UK. J Med Econ. 2014; 17(3): 198–206. PubMed Abstract \| Publisher Full Text\n\nRoggeri DP, Mazzaferro S, Brancaccio D, et al.: Pharmacological control of secondary hyperparathyroidism in hemodialysis subjects: a cost consequences analysis of data from the FARO study. J Med Econ. 2012; 15(6): 1110–17. PubMed Abstract \| Publisher Full Text\n\nRottembourg J, Tilleul P, Deray G, et al.: Cost of managing anemia in end-stage renal disease: the experience of five French dialysis centers. Eur J Health Econ. 2015; 16(4): 357–64. PubMed Abstract \| Publisher Full Text\n\nWilson PD, Hutchings A, Jeans A, et al.: An analysis of the health service efficiency and patient experience with two different intravenous iron preparations in a UK anaemia clinic. J Med Econ. 2013; 16(1): 108–14. PubMed Abstract \| Publisher Full Text\n\nGoodrich NP, Schaubel DE, Smith AR, et al.: National Assessment of Hospitalization Rates for Incident End-Stage Renal Disease After Liver Transplantation. Transplantation. 2016; 100(10): 2115–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGreen JA, Mor MK, Shields AM, et al.: Associations of health literacy with dialysis adherence and health resource utilization in patients receiving maintenance hemodialysis. Am J Kidney Dis. 2013; 62(1): 73–80. PubMed Abstract \| Publisher Full Text\n\nKim Y, Shi J, Freeman CM, et al.: Addressing the challenges of sleeve gastrectomy in end-stage renal disease: analysis of 100 consecutive renal failure patients. Surgery. 2017; 162(2): 358–65. PubMed Abstract \| Publisher Full Text\n\nMinatodani DE, Berman SJ: Home telehealth in high-risk dialysis patients: a 3-year study. Telemed J E Health. 2013; 19(7): 520–2. PubMed Abstract \| Publisher Full Text\n\nMolony JT, Monda KL, Li S, et al.: Effects of Epoetin Alfa Titration Practices, Implemented After Changes to Product Labeling, on Hemoglobin Levels, Transfusion Use, and Hospitalization Rates. Am J Kidney Dis. 2016; 68(2): 266–76. PubMed Abstract \| Publisher Full Text\n\nPark H, Rascati KL, Lawson KA, et al.: Health Costs and Outcomes Associated with Medicare Part D Prescription Drug Cost-Sharing in Beneficiaries on Dialysis. J Manag Care Spec Pharm. 2015; 21(10): 956–64. PubMed Abstract \| Publisher Full Text\n\nWeisbord SD, Mor MK, Sevick MA, et al.: Associations of depressive symptoms and pain with dialysis adherence, health resource utilization, and mortality in patients receiving chronic hemodialysis. Clin J Am Soc Nephrol. 2014; 9(9): 1594–602. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFeldman ZM, Liu LB, Abramowitz SD, et al.: Hemodialysis Vascular Access: Rising Costs as a Surrogate Marker for Patency and Function of Arteriovenous Fistulas. Ann Vasc Surg. 2017; 38: 136–43. PubMed Abstract \| Publisher Full Text\n\nHynes DM, Stroupe KT, Fischer MJ, et al.: Comparing VA and private sector healthcare costs for end-stage renal disease. Med Care. 2012; 50(2): 161–70. PubMed Abstract \| Publisher Full Text\n\nMendu ML, Lundquist A, Aizer AA, et al.: Clinical predictors of diagnostic testing utility in the initial evaluation of chronic kidney disease. Nephrology (Carlton). 2016; 21(10): 851–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPiwko C, Vicente C, Marra L, et al.: The STARRT trial: a cost comparison of optimal vs sub-optimal initiation of dialysis in Canada. J Med Econ. 2012; 15(1): 96–104. PubMed Abstract \| Publisher Full Text\n\nRodby RA, Umanath K, Niecestro R, et al.: Ferric Citrate, an Iron-Based Phosphate Binder, Reduces Health Care Costs in Patients on Dialysis Based on Randomized Clinical Trial Data. Drugs R D. 2015; 15(3): 271–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSibbel S, Sato R, Hunt A, et al.: The clinical and economic burden of pneumonia in patients enrolled in Medicare receiving dialysis: a retrospective, observational cohort study. BMC Nephrol. 2016; 17(1): 199. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSolid CA, Carlin C: Timing of arteriovenous fistula placement and Medicare costs during dialysis initiation. Am J Nephrol. 2012; 35(6): 498–508. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFreeman C, Giles L, Field P, et al.: Supplementary content 5 – Protocol. figshare. http://www.doi.org/10.6084/m9.figshare.11112578\n\nFreeman C, Giles L, Field P, et al.: Supplementary content 6 – PRISMA checklist. figshare. http://www.doi.org/10.6084/m9.figshare.10011893" }	[ { "id": "64135", "date": "11 Jun 2020", "name": "Xue Song", "expertise": [ "Reviewer Expertise Oncology", "autoimmune diseases", "cardiovascular diseases", "chronic kidney disease" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes a systematic literature review on quality of life, symptom burden, and healthcare resource utilization and costs of CKD across different CKD stages and comparing to the general population without CKD. The literature search process, decisions on which publications to include, the summary of review results, and limitations are clearly stated.\nMy only suggestion is to remove the following sentence from Methods, Citation Screening and Full Text Review on page 3 because this information is duplicate from the previous subsection Systemic Literature Review:\n\n\"For the humanistic burden SR, study publication dates for data extraction were restricted to 2007–2017; for the economic burden SR, the relevant time period was restricted to 2012–2017.\"\n\nI recommend acceptance with this minor edit.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] }, { "id": "65877", "date": "09 Jul 2020", "name": "Boris Bikbov", "expertise": [ "Reviewer Expertise chronic kidney disease", "acute kidney disease" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Freeman et al. presents the results of a systematic review on changes in quality of life (QoL) and the economic burden of chronic kidney disease (CKD). The authors performed a search in several databases, collected valuable information and made important conclusions. However, there are several issues that should be improved or further detailed, as explained below:\nIn the “Introduction” the authors refer to a meta-analysis on CKD prevalence suggesting 11-13% of population having the disease. A more recent study on global CKD prevalence is available that estimates the CKD prevalence as substantially lower 9.1% (https://doi.org/10.1016/S0140-6736(20)30045-3).\n\nIt is unclear why the authors limited their systematic search by May 2017.\n\nThe search strategy (described in detail in the supplement at https://figshare.com/articles/Freeman_et_al_Supplementary_content_1_Table_S1/10011476) has a potential pitfall of low sensitivity because it applies exclusion of bibliographic records based on the presence of the word “acute” in a title or an abstract. However, in the abstract of data sources describing the QoL or economy of CKD the word “acute” could occur just to identify that acute kidney diseases were not considered in a given data source. The exact influence of this feature applied by the authors is not known, but it should be discussed in the “Limitations” as a potential reason for lowering search strategy sensitivity, or additional searches should be performed to demonstrate this feature has not led to the exclusion of useful data sources.\n\nThe “Methods” indicates that “Abstracts and titles identified were screened by an independent reviewer”, while the current recommendation is to perform screening by 2 reviewers in order to lower the risk of selection bias or a human error.\n\nThe phrase “Owing to a large number of citations meeting these criteria, a decision was taken to restrict the extraction of data from eligible studies.” in the “Methods” is not clear and should be explained in detail. . The authors screened congress abstracts, but stated that “This meant that, although congress abstract screening was performed, no data from congress abstracts were extracted.” It would be more logical not to mention congress abstracts at all if they have not been used.\n\nIn the inclusion criteria, the authors listed CKD stages from 2 to 5, and it is unclear why they excluded CKD stage 1. They also stated that the inclusion criteria was “eGFR < 75 mL/min/1.73 m2”, but this threshold is very unusual since it does not corresponds neither to KDOQI nor to KDIGO classifications (which use threshold eGFR < 60 mL/min/1.73 m2), and thus the criteria used by the authors identified both healthy persons (with eGFR>60) and CKD patients (with eGFR <60) but excluded persons with eGFR>75 who represent a majority of the general population. The authors identified a list of countries they used as the inclusion/exclusion criteria but this list is not inclusive: it includes some countries of Western Europe but not others, it includes China but not includes Taiwan which has great data on the topic.\n\nAll these methodological pitfalls decrease the value of the review and its results, and should be explained in the “Methods” and discussed in the “Limitations”.\n\nIn the “Results” it is stated that “plus 79 abstracts identified in supplementary searches”, but in the “Methods” the authors indicated they excluded abstracts from the consideration.\n\n“Figure 2” could be presented as a table, without repetition of some rows’ headings.\n\nAt figure 3 the labels “dialysis-dependent CKD” and “dialysis-dependent ESKD” leave some doubts on how the authors use the terminology.\n\nThe authors describe the included studies in the “Results” but perform this in a way that certainly requires improvements. First, it is much better to present all studies in a structured table than in a less-structured text with only some highlights from the included papers. Second, it would be better to group the included papers by whether they consider only pre-dialysis CKD or only ESKD. Neither the main text nor the supplements do not provide enough details of the included papers in this structured way.\n\nThe presentation of “Results” looks more like a listing of the studies but not an exhaustive summary and detailed analysis. I would suggest to put the majority of the extracted data into the tables as described above, and use the “Results” to indicate the most important findings or patterns as already partially done.\n\nThe “Discussion” consists of only the authors’ text and does include only one reference, even without appropriate citation. Actually, the “Discussion” in its current view is more similar to “Conclusion”. In any way, the “Discussion“ should be strengthened.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] }, { "id": "65886", "date": "17 Jul 2020", "name": "Willem Jan W Bos", "expertise": [ "Reviewer Expertise Nephrology", "outcomes of care" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFreeman et al. started an ambitious project by reporting meta-analyses on both the quality of life (QoL) and the cost of treatment of patients with chronic kidney disease (CKD) The overall conclusion confirms existing knowledge that progressing CKD is associated with a reduction in QoL and increasing costs.\nI have several concerns:\nTo describe and analyse the QoL over all stages of kidney disease is a major undertaking. So is meta-analyzing the costs. The authors do not clearly describe why they decided to perform two meta-analyses and report it in one manuscript. In their manuscript, the authors in essence describe two meta-analyses. In clinical practice, and in governing healthcare, it is important to know the effects of a disease and its treatment on both QoL and costs. I miss a clear rationale for why both topics are presented combined in this manuscript. The manuscript reads like a manuscript on QoL and a second on costs. The authors do not make a clear connection between the two. I would invite the authors to better connect the two topics or consider writing two separate manuscripts, one focusing on QoL and one focusing on costs.\n\nThe cut off date used for both analyses was May 2017. Both QoL of kidney patients and costs of kidney care are topics of major interest in recent years. Many studies have been published since. For a manuscript to be published in 2020, an update of the search is essential\n\nThe literature was screened by a single independent reviewer. Reviewing by two reviewers is the accepted norm in meta-analyses.\n\nThe authors do not provide a table stating the risks of bias in the studies analyzed (Prisma checklist item 12)\n\nIn table 2 the authors provide an overview of the literature reported in both meta-analyses. In the first column the text is rotated 90 degrees, making this column illegible (on my laptop). In the current lay-out I can not read which studies are reviewed!\n\nIt is difficult to summarize studies with different ways of staging CKD, using different measurement tools. The authors mainly describe the results of individual studies, both in the figures and in the text of the manuscript. Prisma guidelines suggest providing a summary and a synthesis of results (Prisma items 13,14). Not all studies selected use the standard KDIGO staging of CKD and not all use the same measures of QoL and costs. I would suggest a synthesis of results from those studies that do use the standard KDIGO classification of CKD and do use the same measures of QoL and costs.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-2142
https://f1000research.com/articles/8-2137/v1	23 Dec 19	{ "type": "Software Tool Article", "title": "vhcub: Virus-host codon usage co-adaptation analysis", "authors": [ "Ali Mostafa Anwar", "Mohamed Soudy", "Radwa Mohamed", "Mohamed Soudy", "Radwa Mohamed" ], "abstract": "Viruses show noticeable evolution to adapt and reproduce within their hosts. Theoretically, patterns and factors that affect the codon usage of viruses should reflect evolutionary changes that allow them to optimize their codon usage to their hosts. Some software tools can analyze the codon usage of organisms; however, their performance has room for improvement, as these tools do not focus on examining the codon usage co-adaptation between viruses and their hosts. This paper describes the vhcub R package, which is a crucial tool used to analyze the co-adaptation of codon usage between a virus and its host, with several implementations of indices and plots. The tool is available from: https://cran.r-project.org/web/packages/vhcub/.", "keywords": [ "Evolution", "Natural selection", "Adaptation", "Viruses", "Codon Usage Bias", "R", "RStudio" ], "content": "Introduction\n\nDuring the translation process from mRNAs to proteins, information is transmitted in the form of triple nucleotides, named codons, which encode amino acids. Multiple codons that encode one amino acid are known as synonymous codons. Studies concerning different organisms report that synonymous codons are not used uniformly within and between genes of one genome, a phenomenon known as codon usage bias (CUB)1,2. Since viruses rely on the tRNA pool of their hosts in the translation process, previous studies suggest that translation selection or/and directional mutational pressure act on the codon usage of the viral genome to optimize or deoptimize it towards the codon usage of their hosts3,4.\n\nTools and packages are available to analyze codon usage, e.g. coRdon5, but there is no package available that focuses on the examination of codon usage co-adaptation between viruses and their hosts. vhcub is a package implemented in R, which aims to easily analyze the co-adaptation of codon usage between a virus and its host. vhcub measures several codon usage bias measurements, such as effective number of codons (ENc)6, codon adaptation index (CAI)7, relative codon deoptimization index (RCDI)8, similarity index (SiD)9, synonymous codon usage orderliness (SCUO)10, and relative synonymous codon usage (RSCU)10. It also provides a statistical dinucleotide over- and under-representation with three different models.\n\n\nMethods\n\nvhcub imports Biostrings11, seqinr12 and stringr13 to handle fasta files and manipulate DNA sequences. Also, it imports coRdon5, which is used to estimate different CUB measures.\n\nvhcub first converts the fasta format to data.frame type, to efficiently maintain and calculate different indices implemented in the package. Table 1 describes all the functions available in vhcub, and the result returned from each. Also, it contains references to the equations used to estimate each measure. Furthermore, vhcub uses ggplot214 to visualize two important plots named ENc-GC3 plot (Figure 2) and PR2-plot (Figure 3), which help to explain the factors influencing a virus’s evolution concerning its CUB.\n\nvhcub was developed using R and is available on CRAN. It is compatible with Windows, and major Linux operating systems. The package can be installed as:\n\n\n\nFigure 1 describes the vhcub workflow. It starts with reading the fasta files for a virus and its host. After, nucleotide content analysis, codon usage bias analysis on genes and codon level (marked by the red boxes in Figure 1) can be applied independently (the blue boxes in Figure 1). However, within the same analysis, some measures rely on others. For example, the reference set of genes used to estimate a virus codon adaptation index was defined based on the effective number of codons of its host. Finally, the orange boxes in Figure 1 represent the two plots (ENc-GC3 plot and PR2-plot).\n\nThe white boxes represent the input fasta files. The red boxes represent three main analysis, each with different measures (the blue boxes), and the orange boxes represent ENc-GC3 plot and PR2-plot.\n\n\nUse cases\n\nUsing vhcub to study the CUB of a virus, its host and the co-adaptation between them is straightforward. As an example, we have used the coding sequences for Escherichia virus T4 and its host Escherichia coli in the form of fasta format.\n\n\n\nAs mentioned before, each category of analysis could be applied independently. Hence, this example will show only the codon usage bias analysis at the codon level.\n\n\n\nSiD measures the effect of the codon usage bias of the E. coli on E. coli T4 virus. In general, SiD ranged from 0 to 1 with higher values indicating that the host has a dominant effect on the usage of codons. In this example, SiD is approximately equal to 0.491. Which means that E. coli does not dominate E. coli T4 CUB. Also, this code generates RSCU values for each codon in each gene from both organisms and can be used for further analysis.\n\n\nConclusions\n\nvhcub depends only on DNA sequences as input and can compute different measures of CUB for viruses, such as ENc, CAI, SCUO, and RCDI (Table 1). It can also be used to study the association between viruses and their hosts’ RSCU and SiD. There are many possible directions for future work; further versions will execute more indices, plots, and statistical analysis, to facilitate the workflow for examining the adaptations of viruses’ CUB in the R environment.\n\n\nData availability\n\nEscherichia virus T4 fasta file: ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/836/945/GCF_000836945.1_ViralProj14044\n\nEscherichia coli fasta file: ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/005/845/GCF_000005845.2_ASM584v2/GCF_000005845.2_ASM584v2_cds_from_genomic.fna.gz\n\n\nSoftware availability\n\nSoftware available from: https://CRAN.R-project.org/package=vhcub\n\nSource code available from: https://github.com/AliYoussef96/vhcub\n\nArchived source code as at time of publication: http://doi.org/10.5281/zenodo.357239118\n\nLicense: GPL-3", "appendix": "References\n\nBehura SK, Severson DW: Comparative analysis of codon usage bias and codon context patterns between dipteran and hymenopteran sequenced genomes. PLoS One. 2012; 7(8): e43111. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoël G, Letso R, Neely H, et al.: Codon influence on protein expression in E. coli correlates with mRNA levels. Nature. 2016; 529(7586): 358–363. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurns CC, Shaw J, Campagnoli R, et al.: Modulation of poliovirus replicative fitness in HeLa cells by deoptimization of synonymous codon usage in the capsid region. J Virol. 2006; 80(7): 3259–3272. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCladel NM, Hu J, Balogh KK, et al.: CRPV genomes with synonymous codon optimizations in the CRPV E7 gene show phenotypic differences in growth and altered immunity upon E7 vaccination. PLoS One. 2008; 3(8): e2947. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElek A, Kuzman M, Vlahovicek K: coRdon: Codon Usage Analysis and Prediction of Gene Expressivity. R package version 1.0.3. 2019. Reference Source\n\nNovembre JA: Accounting for background nucleotide composition when measuring codon usage bias. Mol Biol Evol. 2002; 19(8): 1390–1394. PubMed Abstract \| Publisher Full Text\n\nSharp PM, Li WH: The codon Adaptation Index--a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 1987; 15(3): 1281–1295. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPuigbò P, Aragonès L, Garcia-Vallvé S: RCDI/eRCDI: a web-server to estimate codon usage deoptimization. BMC Res Notes. 2010; 3(1): 87. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou JH, Zhang J, Sun DJ, et al.: The distribution of synonymous codon choice in the translation initiation region of dengue virus. PLoS One. 2013; 8(10): e77239. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWan XF, Xu D, Kleinhofs A, et al.: Quantitative relationship between synonymous codon usage bias and GC composition across unicellular genomes. BMC Evol Biol. 2004; 4(1): 19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPagès H, Aboyoun P, Gentleman R, et al.: Biostrings: Efficient manipulation of biological strings. R package version 2.50.2. 2019. Reference Source\n\nCharif D, Lobry JR: SeqinR 1.0-2: a contributed package to the R project for statistical computing devoted to biological sequences retrieval and analysis. In: U. Bastolla, M. Porto, H.E. Roman, and M. Vendruscolo, editors. Structural approaches to sequence evolution: Molecules, networks, populations, Biological and Medical Physics, Biomedical Engineering. Springer Verlag, New York. 2007; 207–232. Publisher Full Text\n\nWickham H: stringr: Simple, Consistent Wrappers for Common String Operations. R package version 1.4.0. 2019. Reference Source\n\nWickham H: ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York. 2016. Reference Source\n\nHe Z, Gan H, Liang X: Analysis of Synonymous Codon Usage Bias in Potato Virus M and Its Adaption to Hosts. In. Viruses. 2019; 11(8). pii: E752. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXiang H, Zhang R, Butler RR 3rd, et al.: Comparative Analysis of Codon Usage Bias Patterns in Microsporidian Genomes. PLoS One. 2015; 10(6): e0129223. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nButt AM, Nasrullah I, Qamar R, et al.: Evolution of codon usage in zika virus genomes is host and vector specific. Emerg Microbes Infect. 2016; 5(10): e107. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoussef A: AliYoussef96/vhcub: Virus-Host Codon Usage Co-Adaptation Analysis (Version v1.0.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3572391" }	[ { "id": "58975", "date": "11 Feb 2020", "name": "Raj Kumar Singh", "expertise": [ "Reviewer Expertise Viral immunology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nViruses in the course of their evolution would optimize their codon usage to their hosts. They rely on the tRNA pool of their hosts in the translation process. Though tools for analyzing the codon usage of organisms are available, none of them focus on examining the codon usage co-adaptation between viruses and their hosts. This software, vhcub, is a tool used to analyze the co-adaptation of codon usage between a virus and its host. This may also help to predict the possible mutations that would accumulate in the virus vis - a - vis its host(s), thereby showing the readiness for the control and prevention of the disease.\nGeneral comments\n1. Corrections in the text\n\nSpelling of formate may be corrected to format in the second column X first row of Table 1 Please define df.fasta In Third column X sixth row and Third column X eight row are one and the same - please explain or correct\nSpecific Whether it can be used in Eukayotes?\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5222", "date": "12 Feb 2020", "name": "Ali Mostafa", "role": "Author Response", "response": "General comments (Corrections in the text) Comment: The spelling of formate may be corrected to format in the second column X first row of Table 1. Response: I will make this correction during the article revisions. Comment: Please define df.fasta Response: (df.host) as well as (df.virus) are just variables names for data frames holds host genes and virus genes, respectively. The definition will be added during the article revisions. Comment: In Third column X sixth row and Third column X eight row are one and the same - please explain or correct Response: In the third column X eight row. It will be corrected from ENc to RCDI. Specific comments Comment: Whether it can be used in Eukaryotes? Response: The translation codon table (The Genetic Codes Tables) number could be changed to any table number (As defined by NCBI https://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi), in vhcub. For example in the function named ( CAI.values() ), one can pass an argument genetic.code=\"11\" for bacterial codon table or genetic.code=\"1\" for eukaryotes. Hence, yes, the host can be Prockayotic or Eukaryotic (vhcub can be used for Eukaryotes)." }, { "c_id": "5233", "date": "13 Feb 2020", "name": "Raj Kumar Singh", "role": "Reviewer Response", "response": "The authors have accepted to do the necessary changes in the revised version and as well they have answered to my query." } ] }, { "id": "61560", "date": "27 Mar 2020", "name": "Oscar Leonardo Ramírez Suárez", "expertise": [ "Reviewer Expertise Virology", "molecular biology", "infectious diseases" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFrom the technical point of view, the vhcub R package looks quite reliable and well supported. However, there is only one example illustrating it. Moreover, this example shows the mean value for SiD in its range (i.e., 0.491 or approx. 0.5), which is great but makes us wondering if this package could give expected values for other cases. If that is possible, could the authors include a couple of examples where the SiD value were below 0.5 and above 0.5?\n\nThe tool is very interesting and captivating. The advantages that R offers are infinite, so I consider that it would be invaluable to exploit the output that R offers. It is clear in the article that the algorithm only allows the entry of DNA sequences in Fasta format, although there are other very simple tools to use to transcribe from RNA to DNA, or from RNA- to RNA + and DNA, it would be very nice to use the same R package to carry out this step, especially considering that from biological tests we not only analyze one sequence but many. To the same extent, I consider that the figures presented in the article should be more discussed from a biological point of view, they could be more informative.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-2137
https://f1000research.com/articles/8-1682/v1	24 Sep 19	{ "type": "Research Article", "title": "A survey exploring biomedical editors’ perceptions of editorial interventions to improve adherence to reporting guidelines", "authors": [ "David Blanco", "Darko Hren", "Jamie J. Kirkham", "Erik Cobo", "Sara Schroter", "Darko Hren", "Jamie J. Kirkham", "Erik Cobo", "Sara Schroter" ], "abstract": "Background: Improving the completeness of reporting of biomedical research is essential for improving its usability. For this reason, hundreds of reporting guidelines have been created in the last few decades but adherence to these remains suboptimal. This survey aims to inform future evaluations of interventions to improve adherence to reporting guidelines. In particular, it gathers editors’ perceptions of a range of interventions at various stages in the editorial process.\n\nMethods: We surveyed biomedical journal editors that were knowledgeable about this topic. The questionnaire included open and closed questions that explored (i) the current practice of their journals, (ii) their perceptions of the ease of implementation and the potential effectiveness of different interventions, (iii) the barriers and facilitators associated with these interventions, and (iv) suggestions for future interventions and incentives. Results: Of the 99 editors invited, 24 (24%) completed the survey. Involving trained editors or administrative staff was deemed the potentially most effective intervention but, at the same time, it was considered moderately difficult to implement due to logistic and resource issues. Participants believed that checking adherence to guidelines goes beyond the role of peer reviewers and could decrease the overall quality of reviews. Journals incentivising adherence, and publishers and medical institutions encouraging journals to adopt strategies to boost adherence were two recurrent themes. Conclusions: Further evaluation of interventions are required. These evaluations could take into account the points raised in this survey.", "keywords": [ "Completeness of reporting", "Journal policies", "Quality of reporting", "Reporting guidelines", "Survey", "Barriers", "Facilitators" ], "content": "Abbreviations\n\nRGs: reporting guidelines; CONSORT: Consolidated Standards of Reporting Trials; RCT: Randomised controlled trials; EQUATOR: Enhancing the QUAlity and Transparency Of Health Research; MiRoR: Methods in Research on Research; STROBE: STrengthening the Reporting of OBservational studies in Epidemiology; PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses; APCs: article processing charges; CME: continuing medical education; ICJME: International Committee of Medical Journal Editors.\n\n\nIntroduction\n\nTransparent and accurate reporting of research is essential for increasing the usability of available research evidence1. Reporting guidelines (RGs) can be useful tools to help authors report research methods and findings in a way that they can be understood by readers, replicated by researchers, used by health care professionals to make clinical decisions, and included in systematic reviews1. Since the inception in 1996 of the Consolidated Standards of Reporting Trials (CONSORT) for the reporting of randomised controlled trials (RCTs)2, more than 400 RGs for different study types, data, and clinical areas have been developed. These RGs can be found in the library of the Enhancing the Quality and Transparency Of Health Research (EQUATOR) Network1.\n\nBiomedical authors’ adherence to RGs has been observed to be suboptimal3. Consequently, in recent years various stakeholders have proposed, and sometimes evaluated, the impact of different types of interventions to improve this adherence. These interventions were identified and classified in a recently published scoping review4. We found that the strategies most widely used by journals have been shown not to have the desired effect5–8 and this highlighted the need for the implementation and evaluation of the other interventions proposed4.\n\nThis paper reports a survey aimed to inform the future evaluation of interventions to improve adherence to RGs. In particular, we focused on interventions that can be implemented at various points in the editorial process. Our specific objectives were to explore the perceived ease of implementation and potential effectiveness of various interventions; to map the barriers and facilitators associated with these interventions; to determine possible solutions to overcome the barriers described, and to identify further editorial interventions that could be implemented and subsequently evaluated.\n\n\nMethods\n\nPurposive sampling was used to recruit biomedical editors that were knowledgeable about the topic we aimed to explore. Participants were sampled from three sources: (i) editors of journals that had published studies describing interventions to improve adherence to RGs identified in our scoping review4, (ii) members of the Methods in Research on Research (MiRoR) Network with current editorial positions and (iii) editors of the top-10 journals (based on impact factor) of BMJ Publishing Group which, apart from being one of the partner institutions of MiRoR, has published the main RGs9–12 and has traditionally performed research to improve the transparency and quality of biomedical publications13. The authors of this survey who met the eligibility criteria were excluded as potential participants.\n\nTo contact editors not known to us we sought email addresses in the public domain. Three editors (including the editors-in-chief) of each of the sampled journals, as well as individual editors from the group (ii) above, were sent a personalised email inviting them to complete an online survey investigating their opinions about different editorial interventions to improve author adherence to RGs. The survey was administered by SurveyMonkey and was open between 27 November 2018 and 24 February 2019. Each survey invitation was tied to a unique email address. Two reminders to complete the survey were sent to non-responders at four and eight weeks after the initial mailing. Participants could edit their responses while completing the survey, but not re-enter the survey once it was completed. We recorded how many people opened the invitation or clicked through to the survey, as well as the number of surveys completed. Participants could suggest further editors that they considered could contribute to the survey. These participants were also sent a personalised invitation.\n\nOur previous scoping review4 identified 31 interventions targeting different stakeholders in the research process. For use in this survey we chose a smaller subset of nine interventions that could be implemented during the editorial process as our focus was on journal editors’ perceptions (see Box 1).\n\nA. Interventions targeting authors:\n\n• A requirement for authors to submit a completed RG checklist (using all appropriate extensions, if applicable) indicating the page numbers where each item is addressed (Intervention 1)\n\n• A requirement for authors to submit a populated RG checklist with text from their manuscript in order to facilitate the peer review process (Intervention 2)\n\n• A requirement for authors to highlight in the manuscript where each RG item is addressed (Intervention 3)\n\n• A requirement for authors to include new subheadings within their manuscript corresponding to different RG items within the traditional IMRaD format (Introduction, Methods, Results, and Discussion) (Intervention 4)\n\n• A requirement for authors on submission to use a freely available writing aid tool that guides authors through the RG checklist items, shows the key elements that need to be reported, and includes examples of adequate reporting (e.g. COBWEB) (Intervention 5)\n\nB. Interventions targeting peer reviewers:\n\n• Instruct peer reviewers to use the appropriate RGs when assessing a manuscript (Intervention 6)\n\n• Instruct peer reviewers to scrutinise the completed RG checklist submitted by the authors and check its consistency with the information reported in the manuscript (Intervention 7)\n\nC. Interventions targeting editorial staff:\n\n• An evaluation of the completeness of reporting by a trained editor (or editorial assistant), who would return incomplete manuscripts to authors before considering the manuscript for publication (Intervention 8)\n\nD. Interventions targeting authors, peer reviewers, and editors:\n\n• Training for authors, peer reviewers, and editors on the importance, content, and use of RGs (e.g. The EQUATOR Network toolkits) (Intervention 9)\n\nThe survey combined open and closed response questions to seek participants’ perceptions of a series of interventions to improve authors’ adherence to RGs that could potentially be implemented during the editorial process. We pilot tested the draft survey questionnaire with two collaborators of the MiRoR project who currently hold editorial positions. They were asked to review the survey for its clarity and completeness and to provide suggestions on how to improve its structure.\n\nBased on feedback from the pilot we decided not to include the intervention “Implementation of the automatic tool Statreviewer14” since participants were not aware of this software and stated that their perceptions would strongly depend on details about how it operates which are not publicly available.\n\nWe structured the final questionnaire (see Figure S1, Extended data)15 as follows:\n\nPart 1: Current practice. Participants were asked to describe the measures their journal currently takes to improve adherence to RGs.\n\nPart 2: Perceptions of nine potential interventions. Participants were asked to indicate on 5-point Likert scales (i) how easy it would be (or was) to implement these interventions at their journals (1-very difficult, 2-moderately difficult, 3-neither difficult nor easy, 4-moderately easy, 5-very easy) and (ii) how effective they thought the interventions would be (or was) at improving adherence to RGs if these were implemented at their journals (1-very ineffective, 2-moderately ineffective, 3-neither ineffective nor effective, 4-moderately effective, 5-very effective). We included images to clarify meanings and context to prompt participants to think about the benefits and drawbacks of the interventions. Free text boxes were included so participants could justify their responses.\n\nPart 3: Identifying the barriers and facilitators. Participants were asked to choose which intervention they considered potentially the most effective for their journal. They were asked to describe (i) why they thought that intervention would be the most effective, (ii) what the main difficulties in implementing that intervention would be, and (iii) how they would try to overcome these difficulties.\n\nPart 4: Further interventions. Participants were asked for further suggestions of possible interventions, including modifications and combinations of the interventions previously discussed.\n\nPart 5: Demographic questions.\n\nDescriptive statistics were calculated for quantitative data using R version 3.6.016. For qualitative information, the lead investigator (DB) used the software program NVivo 1217 to extract data and classify the data into key themes. This classification was discussed with another investigator (SS) and subsequently refined.\n\nFor Part 1 of the survey (Current practice) the unit of measure were the journals and therefore editors of the same journal were grouped. For all other parts of the survey, we analysed editors’ responses independently, no matter what their journal was.\n\nThe Research Committee of the Governing Council of the Universitat Politècnica de Catalunya (UPC) granted ethical approval for this study (Reference EC 01, Date 2 May 2018).\n\nParticipants were informed that completion of the survey indicated consent to participate, and that they were free to stop and withdraw from the study at any time without providing a reason.\n\nWe consulted the Checklist for Reporting of Results of Internet E-Surveys (CHERRIES)18 and the Consolidated criteria for Reporting of Qualitative research (COREQ)19 guidelines to produce this research report.\n\n\nResults\n\nOf the 99 editors invited, 42 opened the invitation (view rate 42%), and 24 completed the survey (response rate 24%) from the 25 who started it (completion rate 96%). The average time spent completing the survey was 15 minutes (SD = 8.5 minutes). The 24 participants were editors of 20 different biomedical journals and had a variety of editorial roles (editor-in-chief, senior editor, associate editor or others). Most of them were involved in manuscript decision-making and had less than 15 years of experience as journal editors. Table 1 shows their demographic characteristics. Raw survey results are given as Underlying data20.\n\nRespondents worked at 20 journals. Most respondents’ journals (12/20, 60%) request authors to submit a completed RG checklist with page numbers indicating where the items are addressed when they submit their manuscript. A further seven (35%) instruct but do not request authors to do it, and one (5%) does not request or instruct authors. Among the journals requesting the submission of checklists, four (4/12, 33%) also explicitly ask peer reviewers to use the completed RGs when assessing manuscripts, one (1/12, 8%) asks peer reviewers general questions about the completeness of reporting, and one performs an evaluation of the completeness of reporting by a trained editor using RGs before the initial decision is made on the paper. We observed no incongruences between the answers of editors from the same journal. Some respondents mentioned that in their journals (n=4) the interventions described were only applicable to the study types corresponding to the most established RGs (CONSORT, STROBE, or PRISMA) for trials, observational studies and systematic reviews respectively.\n\nThe mean scores for perceived ease of implementation and potential effectiveness for each intervention are shown in Figure 1.\n\nWhite dots represent the mean scores for each of the categories. Interventions in red target authors, those in grey target peer reviewers, the one in orange target editors or administrative staff and the one in blue targets all these stakeholders. An explanation of the content of each intervention can be found in Box 1.\n\nThe two most common interventions were considered the easiest ones to implement: the mean scores for requesting authors to submit checklists with page numbers (Intervention 1) and for asking peer reviewers to use RGs (Intervention 6) were 4.33 (SD=0.90) and 3.67 (SD=1.14), respectively. By contrast, interventions related to training (Intervention 9), editor involvement in checking completeness of reporting (Intervention 8) and reformatting of the text based on RG requirements (Intervention 4, Intervention 5) were considered the most difficult to implement.\n\nAn evaluation of the completeness of reporting by a trained editor was considered the most effective intervention (4.09, SD=1.02) and the two targeting peer reviewers (Interventions 6 and 7) were perceived as being the least effective (3.13, SD=1.17; 2.96, SD=1.06). All interventions targeting authors (Interventions 1-5) and training (Intervention 9) ranged between 3.3 and 3.6.\n\nThis section presents the perceived barriers and facilitators of the interventions considered and editors’ suggestions for making the interventions more effective. Table S1 in Extended data15 shows a full description of these.\n\nA) Interventions targeting authors (1-5)\n\nThe main barriers associated with all of the interventions targeting authors was that authors have to state their adherence to the relevant RG and this does not equate to actual compliance. Moreover, it is resource intensive for journals to check that these requirements are appropriately met by authors. Some editors highlighted that Interventions 3, 4, and 5 would involve special formatting of the submitted manuscript, which could be cumbersome for authors given that manuscripts are often submitted to multiple journals with different formats before being accepted. This is particularly relevant for journals with high rejection rates as it could cause frustration for authors. Some participants mentioned logistical issues as their journal’s manuscript tracking system is not set up to accommodate these interventions. In addition, changes in the manuscript’s format could be incompatible with the journal’s house style.\n\nIntervention 1 was generally considered quick and straightforward for authors, but several participants indicated that there is published empirical evidence of little effectiveness if the checklist is not assessed by a trained editor or administrator5–8.\n\nAs Interventions 3, 4, and 5 force authors to tailor the manuscript to RG requirements, participants reported that these could make editors’ and peer reviewers’ jobs easier as the manuscript would be better structured. Importantly, readers would also be able to locate information more easily. Some editors pointed out that, to make these interventions effective, journals would need to provide templates to authors or to integrate these interventions in the submission system. However, some of these interventions (Interventions 2 and 5) were seen as more effective if they were implemented earlier on in the research process, prior to writing the manuscript.\n\nB) Interventions targeting peer reviewers (6, 7)\n\nMost respondents were negative about the potential effectiveness of implementing the two interventions targeting peer reviewers (Intervention 6 and 7) as they felt these would create too much additional work for reviewers. Participants were concerned that the quality of peer review could be compromised as reviewers should focus on the manuscript’s content and not on the reporting issues. Furthermore, peer reviewers may not know which RGs to use and, even if they do, the effectiveness would be dependent on their willingness to use RGs and their expertise in applying them. Several participants indicated that this work should be delegated to paid editorial staff.\n\nC) Interventions targeting editorial staff (8)\n\nThis intervention was considered difficult to implement but potentially effective. The main facilitating factor for its successful implementation was that it is performed by a paid or trained professional, which lends credibility to the intervention, reduces the workload of unpaid peer reviewers, and avoids authors overclaiming adherence. The main barriers outlined for this intervention were (i) the budget issues the journal would need to face to train or hire additional editorial staff that could perform the evaluation, especially if the journal receives a large volume of papers, (ii) the editorial delays it may cause, and the (iii) the potential inefficiency of assistant editors or administrators having to delegate decisions in case of doubt, given that sometimes assessing completeness of reporting is a subjective task.\n\nTo make this intervention more feasible for journals, editors suggested that the completeness of reporting evaluation could be performed only for papers that are sent out for peer review and, it could be focused on a few core items (different for each RG) that would enable reproducibility. If this intervention was implemented in a journal that requires the submission of a completed checklist, editors could take advantage of the checklist to locate information.\n\nD) Interventions targeting authors, peer reviewers and editors (9)\n\nTraining was seen as a potentially effective intervention but difficult to implement. Some participants highlighted that training with follow up sessions would be resource intensive for journals, and especially difficult to enforce. One participant mentioned that credits (such as CME credits21) could be used to recognise hours of training. The fact that sometimes the editorial staff is based in different places and zones makes it crucial to consider flexible forms of training, such as online courses. As an example, the EQUATOR Network Toolkits section provides resources for authors, peer reviewers and journal editors22. However, some participants emphasised that training should also be delivered by research institutions and medical centres.\n\nThe implementation of reading tools that automatically assess adherence to RGs, such as Statreviewer14, were seen as potentially interesting interventions. Some respondents also mentioned the possibility of combining some of the interventions listed, such as requiring the submission of checklists and trained editors assessing the responses with the information reported in the manuscript.\n\nMoreover, several incentives for authors were listed, including (i) discounts on article processing charges (APCs) for authors that comply with RG requirements, (ii) academic institutions including RG use in the promotion and tenure files, and (iii) credits (such as CME credits21) to recognise hours of training on the use of RGs. Journals could also be encouraged to implement certain interventions if (i) there is empirical evidence that these interventions actually improve the reporting quality of the papers or (ii) publishers or the International Committee of Medical Journal Editors (ICMJE) mandate these as a condition of submission to their journals. Even if some of these interventions are proven to be effective, some respondents reported that it is essential to convince publishers that improving the quality of reporting is a worthy investment to resource.\n\n\nDiscussion\n\nThis survey explores biomedical journal editors’ perceptions of the practical aspects of the implementation of different interventions to improve adherence to RGs.\n\nSeveral messages arise from this study. First of all, most editors agreed that the most effective way to improve adherence to RGs is for journals to involve trained editors or administrative staff. Interventions targeting these stakeholders were considered to be difficult to implement for most journals, either because of logistic or resource issues. However, improving the performance of editorial staff is critical23 and has been shown to have a positive impact on completeness of reporting in the context of a dentistry journal24. To make these type of interventions more feasible, journals could implement them only for manuscripts that are sent out for peer review. The editorial staff could also take advantage of the RG checklists submitted by authors, that could be automatically populated with text using specific software such as the the tool proposed by Hawwash et al.25\n\nMost editors considered that checking reporting issues is beyond the role of peer reviewers. Given the voluntary nature of peer review, requiring reviewers to use RGs causes an additional workload that could compromise the overall quality of the reviews. Furthermore, as finding peer reviewers is becoming increasingly difficult for editors26, these requirements could make them even less willing to review papers. Additionally, some editors considered that the average peer reviewer does not have enough expertise to go over RG requirements.\n\nWe observed that the interventions perceived as potentially most effective appear to be more difficult to implement. Conversely, the most common strategies seem to have been implemented based on their feasibility and not on their potential to improve completeness of reporting. This could be one of the reasons why they have failed to achieve the desired results5–8. Some of our respondents insisted that a key element is that journals, universities, and medical institutions find ways to incentivise author’s compliance with RGs. At the same time, the scientific community needs to find ways to convince publishers that improving the quality of reporting is a worthy investment so that publishers can encourage their journals to adopt strategies to boost completeness of reporting. A recent article indicates that implementing RGs through the editorial process may increase the number of citations to the research reported27.\n\nA common observation by the survey participants was that the effectiveness of the interventions proposed could depend on the types of articles considered. While RGs for randomised trial protocols, randomised trials or systematic reviews are more established, some others, including most RG extensions, are not well known to the stakeholders involved in the publication process. For this reason, it is important for journals to be clear in their “Instructions for Authors” on what RGs they mandate.\n\nWe encourage researchers to perform further evaluations of interventions in collaboration with biomedical journals, such as the RCT our research team is currently undergoing28. Our study aims to evaluate the effect on completeness of reporting of a trained researcher assessing during peer review the consistency between the CONSORT checklists submitted by authors and the information reported in the manuscript, and providing authors with a report indicating any inconsistencies found.\n\nProviding high quality evidence of the effectiveness of different interventions to improve adherence to RGs and discussing how to make them less burdensome are key aspects needed to convince all stakeholders that this effort is worth it.\n\n\nData availability\n\nZenodo: Underlying data of the project “A survey exploring biomedical editors’ perceptions of editorial interventions to improve adherence to reporting guidelines”. DOI: https://doi.org/10.5281/zenodo.340772520.\n\nThis project contains the following underlying data:\n\nSurvey dataset (Dataset including all survey responses).\n\nZenodo: Extended data of the project “A survey exploring biomedical editors’ perceptions of editorial interventions to improve adherence to reporting guidelines”. https://doi.org/10.5281/zenodo.340400215.\n\nThis project contains the following extended data:\n\nFigure S1: Survey questionnaire (Complete version of the survey questionnaire used in this project)\n\nTable S1: Barriers, facilitators and possible improvements of the included interventions (Table containing the barriers, facilitators and possible improvements identified for each of the interventions explored in the survey)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nThe authors thank the MiRoR Project and Marie Sklodowska-Curie Actions for their support. This survey is the second part of a larger project whose first part was a scoping review to identify and classify interventions to improve adherence to RGs4. The third part is an RCT to evaluate the impact of assessing during peer review the CONSORT checklist submitted by authors28.\n\n\nReferences\n\nEQUATOR Network: EQUATOR Network website-the resource centre for. 2013. Reference Source\n\nBegg C, Cho M, Eastwood S, et al.: Improving the quality of reporting of randomized controlled trials. The CONSORT statement. JAMA. 1996; 276(8): 637–9. PubMed Abstract \| Publisher Full Text\n\nSamaan Z, Mbuagbaw L, Kosa D, et al.: A systematic scoping review of adherence to reporting guidelines in health care literature. J Multidiscip Healthc. 2013; 6: 169–188. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlanco D, Altman D, Moher D, et al.: Scoping review on interventions to improve adherence to reporting guidelines in health research. BMJ Open. 2019; 9(5): e026589. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShamseer L, Stevens A, Skidmore B, et al.: Does journal endorsement of reporting guidelines influence the completeness of reporting of health research? A systematic review protocol. Syst Rev. 2012; 1(1): 24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStevens A, Shamseer L, Weinstein E, et al.: Relation of completeness of reporting of health research to journals' endorsement of reporting guidelines: systematic review. BMJ. 2014; 348: g3804. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlanco D, Biggane AM, Cobo E, et al.: Are CONSORT checklists submitted by authors adequately reflecting what information is actually reported in published papers? Trials. 2018; 19(1): 80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHair K, Macleod MR, Sena ES, et al.: A randomised controlled trial of an Intervention to Improve Compliance with the ARRIVE guidelines (IICARus). Res Integr Peer Rev. 2019; 4(1): 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchulz KF, Altman DG, Moher D, et al.: CONSORT 2010 statement: updated guidelines for reporting parallel group randomised trials. BMJ. 2010; 340: c332. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. BMJ. 2009; 339: b2535. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvon Elm E, Altman DG, Egger M, et al.: Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. BMJ. 2007; 335(7624): 806–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChan AW, Tetzlaff JM, Gøtzsche PC, et al.: SPIRIT 2013 explanation and elaboration: guidance for protocols of clinical trials. BMJ. 2013; 346: e7586. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEvidence based publishing. Reference Source\n\nStatreviewer. Reference Source\n\nBlanco D: Extended data of the project \"A survey exploring biomedical editors' perceptions of editorial interventions to improve adherence to reporting guidelines\" [Data set]. Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3404002\n\nR: A language and environment for statistical computing. 2019. Reference Source\n\nNVivo qualitative data analysis software. 2018. Reference Source\n\nEysenbach G: Improving the quality of Web surveys: the Checklist for Reporting Results of Internet E-Surveys (CHERRIES). J Med Internet Res. 2004; 6(3): e34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTong A, Sainsbury P, Craig J: Consolidated criteria for reporting qualitative research (COREQ): a 32-item checklist for interviews and focus groups. Int J Qual Heal Care. 2007; 19(6): 349–57. PubMed Abstract \| Publisher Full Text\n\nBlanco D: Underlying data of the project \"A survey exploring biomedical editors' perceptions of editorial interventions to improve adherence to reporting guidelines\" [Data set]. Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3407725\n\nHealth NI of: What is CME Credit? Reference Source\n\nEQUATOR Network Toolkits. Reference Source\n\nCobo E, González JA: Taking advantage of unexpected WebCONSORT results. BMC Med. 2016; 14(1): 204. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPandis N, Shamseer L, Kokich VG, et al.: Active implementation strategy of CONSORT adherence by a dental specialty journal improved randomized clinical trial reporting. J Clin Epidemiol. 2014; 67(9): 1044–8. PubMed Abstract \| Publisher Full Text\n\nHawwash D, Lachat C, De Cock N, et al.: Integrating a writing aid to facilitate the use of reporting guidelines: A crossover randomized controlled trial Trial protocol. Reference Source\n\nPublons: Global State of Peer Review 2018. Reference Source\n\nVilaró M, Cortés J, Selva-O’Callaghan A, et al.: Adherence to reporting guidelines increases the number of citations: the argument for including a methodologist in the editorial process and peer-review. BMC Med Res Methodol. 2019; 19(1): 112. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlanco D, Schroter S, Moher D, et al.: Evaluating the Impact of Assessing During Peer Review the CONSORT Checklist Submitted by Authors. Reference Source" }	[ { "id": "55890", "date": "11 Nov 2019", "name": "Dennis W. Lendrem", "expertise": [ "Reviewer Expertise statistics", "modelling", "validation", "decision making" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper reports on the views of a sample of biomedical editors on editorial interventions to improve adherence to reporting guidelines. The paper is clear, transparent, and well documented.\nA limitation of the study is that the results are based upon a survey return rate of just 24%. The returns are likely biased favouring editors with stronger views on reporting guidelines. In addition, it is not clear whether all ten of the top-ten journals were represented, or whether one or more journals dominate the response rate. In addition it is not completely clear how many participants came from the MiRoR Network, or journals previously publishing studies on Reporting Guidelines.\nFigure 1 is hard work to interpret and might be presented more usefully as two separate graphs. In addition, the colour scheme could be changed to highlight those interventions considered \"Very easy\" or \"Moderately easy\" to implement, and those ranked as \"Very effective\" or \"Moderately effective\". (Note that the proportions of each score could still be retained.)\n\nHowever, this is a useful addition to the literature offering valuable insight into some views on guidelines and candidate interventions promoting closer adherence to guidelines. The paper would be improved by a paragraph on Limitations of the Study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5090", "date": "12 Dec 2019", "name": "David Blanco", "role": "Author Response", "response": "We thank you for your time and comments on the manuscript. Please find below a point by point response to them:A limitation of the study is that the results are based upon a survey return rate of just 24%. The returns are likely biased favouring editors with stronger views on reporting guidelines.We have now reflected on this point in the Discussion section. In addition, it is not clear whether all ten of the top-ten journals were represented, or whether one or more journals dominate the response rate. In addition it is not completely clear how many participants came from the MiRoR Network, or journals previously publishing studies on Reporting Guidelines.We have now added that information to the first paragraph of the Results section. Figure 1 is hard work to interpret and might be presented more usefully as two separate graphs. In addition, the colour scheme could be changed to highlight those interventions considered \"Very easy\" or \"Moderately easy\" to implement, and those ranked as \"Very effective\" or \"Moderately effective\". (Note that the proportions of each score could still be retained.)In order to make Figure 1 easier to interpret, we have substituted the bar plot by a box plot. We have also plotted the individual and mean scores for each intervention and each outcome. Everything is explained in the legend. However, we prefer not to separate it into two graphs as we believe it is interesting for the readers to be able to compare the scores that each intervention obtains for each of the two outcomes.The paper would be improved by a paragraph on Limitations of the Study.We have now included a section on Limitations of the study in the Discussion section." } ] }, { "id": "55889", "date": "20 Nov 2019", "name": "Gerard Urrutia", "expertise": [ "Reviewer Expertise Systematic reviews" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and original study that deepens the knowledge about interventions aimed at improving adherence to reporting guidelines by medical journals, an important issue. The study focuses on the perspective of the biomedical editors and provides valuable information on their perceptions of various interventions that have been proposed, identifies barriers and facilitators and provides possible solutions.\nHowever, the study includes a limited sample of participants (24 editors belonging to 20 medical journals), which limits the scope of the conclusions. Although the study, according to its stated objectives, does not intend to offer a representative view of the editors of all medical journals, the limited number of participants as well as their representativeness (the sources from which they were identified and selected allows us to think that it is a highly selected sample of publishers with previous experience and a potential motivation in the subject), is not mentioned in the discussion. It is noteworthy that, despite this potentially \"favourable\" sample, the response rate is very low (24%). What would have been the participation rate and the responses if a larger and more representative sample had been selected? Although this was not an objective of the study, it would deserve some reflection in the discussion.\nIt would also be useful to provide details on the origin of the participants in the survey according to the sources of selection, which would improve the description of the sample.\nThe results presented both in the section on current practices as well as in perceptions and in barriers and facilitators are consistent, clear and practical. The analysis is descriptive and does not pose difficulties.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5092", "date": "12 Dec 2019", "name": "David Blanco", "role": "Author Response", "response": "We thank you for your time and comments on the manuscript. Please find below a point by point response to them: However, the study includes a limited sample of participants (24 editors belonging to 20 medical journals), which limits the scope of the conclusions. Although the study, according to its stated objectives, does not intend to offer a representative view of the editors of all medical journals, the limited number of participants as well as their representativeness (the sources from which they were identified and selected allows us to think that it is a highly selected sample of publishers with previous experience and a potential motivation in the subject), is not mentioned in the discussion. We have now discussed these points in the Discussion section. It is noteworthy that, despite this potentially \"favourable\" sample, the response rate is very low (24%). What would have been the participation rate and the responses if a larger and more representative sample had been selected? Although this was not an objective of the study, it would deserve some reflection in the discussion. We have now included some comments about the response rate in a new paragraph on Limitations (Discussion section). It would also be useful to provide details on the origin of the participants in the survey according to the sources of selection, which would improve the description of the sample. We have now added that information to the first paragraph of the Results section." } ] }, { "id": "56388", "date": "26 Nov 2019", "name": "Patricia Logullo", "expertise": [ "Reviewer Expertise Methodology. Reporting quality. Research integrity." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPositive points:\n\nThis is a much-needed survey, and I am happy and thankful that the MiRoR team invested their time and expertise in undertaking it. The subject of the adoption of reporting guidelines in the editorial process is sensitive, relevant and worth exploring. Hearing editors is a crucial step for the development of interventions aimed to improve the quality of biomedical research reporting. I congratulate the authors for the initiative.\n\nThe main result of the study (that the interventions perceived as effective are the most difficult to implement and vice-versa) points out to the need to verifying feasibility before proposing interventions, something that we should all think about.\n\nI hope the authors find these suggestions helpful and constructive, and that they find these to be positive contributions.\n\nMajor concerns: methodology\n\n1) Sample characteristics. Although the authors should be praised for the idea of asking editors about interventions to improve reporting, I have a concern about what editors should be surveyed. If one wants to explore the perceived ease of the implementation of one intervention, is it adequate to choose a population of editors whom we know are already “knowledgeable about the topic”?\n\nIf these editors are aware of the reporting quality crisis and the reporting guidelines in general, wouldn’t these be more prone to adopt interventions that aim to improve the quality of publications? Therefore, aren’t these the people who would say “yes” to any intervention proposed?\n\nShould the authors not interview editors who have never heard about reporting guidelines too? Wouldn’t this heterogeneous sample be more representative of the scenario of difficulties — or the ease — of implementing reporting guidelines checks in the editorial process? Are the study results only applicable to journals that already endorse or require reporting guidelines?\n\nI would like to see comments about this limitation in the discussion if the authors agree with this or even if they do not.\n\n2) Sample size. My second most important concern is about sample size. In the Methods section, the authors do not explain how they planned the sample size and based on what assumptions. 24% seems to be a very low response rate. Do the authors believe 24 is enough? This limitation should be discussed.\n\n3) Effectiveness. When you asked your participants about their opinions on “how effective” something would be, what was the definition of effectiveness? Would it be their perception about the intervention improving the adherence to reporting guidelines checklists? Was it a more subjective view of “manuscript quality”?\n\n4) Statreviewer. The authors mention in the text, in Methods: “Based on feedback from the pilot we decided not to include the intervention “Implementation of the automatic tool Statreviewer14” since participants were not aware of this software and stated that their perceptions would strongly depend on details about how it operates which are not publicly available.” However, on page 7, Results, they state: “The implementation of reading tools that automatically assess adherence to RGs, such as Statreviewer14, were seen as potentially interesting interventions.”\n\nAfter all, was this intervention Statreviewer evaluated (as in Results) or was it not (as in Methods)? Do you mean they mentioned other tools, such as Penelope, GoodReports, Cobweb or others? Or were they referring to Statreviewer?\n\nMajor concerns: reporting\n\n5) Journals. You interviewed 24 editors from 20 journals. What are these journals? I did not see a list of their names, and I believe it is important.\n\n6) Figure 1. Figure 1 is difficult to read/interpret. I like the idea of having the two outcomes (ease of implementation and effectiveness) in one figure very much so that one can compare the two. However, the bar chart is not clear at all, nor easy to interpret. Colours do not help. The authors should consider building a figure with plot points (which would be easy, given the small sample) showing where each participant sits within the Likert scale — not bars.\n\nAnother suggestion for Figure 1 (here, just a suggestion) is to add something to remind the readers what the interventions are. As Box 1 is far from the Figure, it is difficult to remember what each intervention is. It would be useful to have “nicknames”, for example: 1. Page numbers 2. Checklist 3. Highlight 4. Subheadings 5. COBWEB 6. Use RG 7. Check RG 8. Staff 9. Training\n\n7) Peer reviewers’ checks. The abstract is, in general, well written and clear. However, I got intrigued by this sentence: “Participants believed that checking adherence to guidelines goes beyond the role of peer reviewers and could decrease the overall quality of reviews.”\n\nI was surprised that peer reviewers checking reporting guidelines adherence would decrease the overall quality of the paper. Then I reread it in the paper, but here put in a complete sentence, that makes sense:\n\n“Participants were concerned that the quality of peer review could be compromised as reviewers should focus on the manuscript’s content and not on the reporting issues.”\n\nMeaning, I suppose, that peer reviewers would focus on checking reporting guidelines and not on their “expert view” of the study. I get it, but it is not very clear, neither in the text, nor in the abstract, and I invite the authors to rewrite both. Especially because “reporting guidelines” are about content too. They point out the content elements that should not be absent from papers, so using the word “content” in the manuscript text is not really appropriate. Please, re-evaluate.\n\n8) As I said above, the abstract is good, and there is room for growth (you have now 232 words). However, I could not find the conclusions in the abstract helpful. Could you please inform better what you did conclude? What “points raised in the survey” should be considered?\n\n9) Checklists as forms. ...“assessing completeness of reporting is a subjective task”. This is one of the most important sentences in this manuscript, it tells us about using reporting guidelines checklists as evaluation tools — for what they have not been created. I saw no discussion about this in your paper.\n\n10) Discussion. The discussion of the paper is short, and it lacks several points pointed above. Mainly, it does not discuss the limitations and potential bias of this study. Discussing limitations is a requirement from F1000Research journal.\n\n11) You mention you did analyse qualitative information using NVivo. How did you do it?\n\n12) There are at least eight items from the CHERRIES checklist that I could not see in your paper, and you might want to report, even if in a separate (or supplementary) box, from data protection, preventing multiple entries, to randomisation of items, and others.\n\n13) From Box 1, I do not quite get the difference between Interventions 1 and 2. It seems that both ask authors to submit a populated/completed RG checklist. Is it that #1 asks for page numbers and #2 does not?\n\n14) In my opinion, the sentences from “To contact editors...” to “improve adherence to RGs” belong more to the “Participants” section than to the “Procedure”. Especially the first sentence. Also “Participants could suggest further editors that they considered could contribute to the survey” is something that I would consider as a snowballing or recruitment method, not the survey procedure per se. So, I would suggest putting everything related to recruitment together in one section, to make it easier for your reader (who might be interested in undertaking a similar survey) to find information on such a critical issue as recruitment. Also: how could participants suggest? Was it a specific question in the SurveyMonkey form?\n\n15) Were participants given an estimation of the time required to answer the survey before they began?\n\nMinor: about citations\n\n16) Reference number 1 (EQUATOR Network website) is not a proper, complete reference. It points to the home page of the website, not to a document, and as so, it does not show from where the authors collected that information (the reader cannot find them in the home page). The correct way of citing would be adding the full URL. A suggestion: EQUATOR Network. What is a reporting guideline? Available at: http://www.equator-network.org/about-us/what-is-a-reporting-guideline/. Cited on 19/11/2019.\n\n17) Something similar happens to reference number 13. Author and title are missing. It would help readers if it could be typed like this: Schoter S. Evidence-based publishing. BMJ. Available at: https://www.bmj.com/about-bmj/evidence-based-publishing. Cited on 19/11/2019.\n\n18) Reference 14 also lacks authors and titles. References 13, 14, 16, 21 and 22 lack a lot of elements necessary for readers to be able to find them. When working in a printed version (as I am now), you cannot see what the document is, where it is published or by whom — these are available only by those reading online and able to “click” on the links.\n\n19) I would change the sentence: “Raw survey results are given as Underlying data20.” To something clearer and that does not require the reader to go to the reference list to understand what it is. “Table XX in the supplementary file X20 shows the responses from all 24 participants.”\n\n20) Has the study by Hawwash et al. (reference 25) been published yet? (The authors cited the protocol only). Do the authors plan to cite other electronic tools that are out there?\n\nThank you for the opportunity of reading and evaluating this paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5094", "date": "12 Dec 2019", "name": "David Blanco", "role": "Author Response", "response": "We thank you for your time and detailed comments on the manuscript. Please find below a point by point response to them:Major concerns: methodology1) Sample characteristics. Although the authors should be praised for the idea of asking editors about interventions to improve reporting, I have a concern about what editors should be surveyed. If one wants to explore the perceived ease of the implementation of one intervention, is it adequate to choose a population of editors whom we know are already “knowledgeable about the topic”? If these editors are aware of the reporting quality crisis and the reporting guidelines in general, wouldn’t these be more prone to adopt interventions that aim to improve the quality of publications? Therefore, aren’t these the people who would say “yes” to any intervention proposed? Should the authors not interview editors who have never heard about reporting guidelines too? Wouldn’t this heterogeneous sample be more representative of the scenario of difficulties — or the ease — of implementing reporting guidelines checks in the editorial process? Are the study results only applicable to journals that already endorse or require reporting guidelines? I would like to see comments about this limitation in the discussion if the authors agree with this or even if they do not.Firstly, we have now included some more information in the “Participants” section to make it clear what kind of participants we were aiming to reach. Secondly, we have discussed in the Discussion section the other points you mention. 2) Sample size. My second most important concern is about sample size. In the Methods section, the authors do not explain how they planned the sample size and based on what assumptions. 24% seems to be a very low response rate. Do the authors believe 24 is enough? This limitation should be discussed.We have now discussed these points in the Discussion section. 3) Effectiveness. When you asked your participants about their opinions on “how effective” something would be, what was the definition of effectiveness? Would it be their perception about the intervention improving the adherence to reporting guidelines checklists? Was it a more subjective view of “manuscript quality”?Thanks to your comment, we have just realised that we only mentioned that we were talking about “effectiveness at improving adherence to RGs” in the methods section, but not in the abstract and introduction. We have now clarified that in the abstract and introduction, and also in some parts of the methods, results and discussion to remind the readers that we refer to effectiveness at improving adherence to RGs. 4) Statreviewer. The authors mention in the text, in Methods:“Based on feedback from the pilot we decided not to include the intervention “Implementation of the automatic tool Statreviewer14” since participants were not aware of this software and stated that their perceptions would strongly depend on details about how it operates which are not publicly available.”However, on page 7, Results, they state:“The implementation of reading tools that automatically assess adherence to RGs, such as Statreviewer14, were seen as potentially interesting interventions.” After all, was this intervention Statreviewer evaluated (as in Results) or was it not (as in Methods)? Do you mean they mentioned other tools, such as Penelope, GoodReports, Cobweb or others? Or were they referring to Statreviewer?We have clarified in the results section (“Further interventions and incentives for authors and journals”) that Statreviewer was not included in the survey but mentioned by some participants when asked about further potentially effective interventions. Major concerns: reporting5) Journals. You interviewed 24 editors from 20 journals. What are these journals? I did not see a list of their names, and I believe it is important.The 20 journals represented are now listed in Table 1. We have also improved the description of the sample in the first paragraph of the Results section. 6) Figure 1. Figure 1 is difficult to read/interpret. I like the idea of having the two outcomes (ease of implementation and effectiveness) in one figure very much so that one can compare the two. However, the bar chart is not clear at all, nor easy to interpret. Colours do not help. The authors should consider building a figure with plot points (which would be easy, given the small sample) showing where each participant sits within the Likert scale — not bars.We have substituted the bar plot by a box plot. We have also plotted the individual and mean scores for each intervention and each outcome. Everything is explained in the legend. Another suggestion for Figure 1 (here, just a suggestion) is to add something to remind the readers what the interventions are. As Box 1 is far from the Figure, it is difficult to remember what each intervention is. It would be useful to have “nicknames”, for example:1. Page numbers2. Checklist3. Highlight4. Subheadings5. COBWEB6. Use RG7. Check RG8. Staff9. TrainingWe have incorporated similar nicknames to help readers remember the interventions. 7) Peer reviewers’ checks. The abstract is, in general, well written and clear. However, I got intrigued by this sentence:“Participants believed that checking adherence to guidelines goes beyond the role of peer reviewers and could decrease the overall quality of reviews.” I was surprised that peer reviewers checking reporting guidelines adherence would decrease the overall quality of the paper. Then I reread it in the paper, but here put in a complete sentence, that makes sense: “Participants were concerned that the quality of peer review could be compromised as reviewers should focus on the manuscript’s content and not on the reporting issues.” Meaning, I suppose, that peer reviewers would focus on checking reporting guidelines and not on their “expert view” of the study. I get it, but it is not very clear, neither in the text, nor in the abstract, and I invite the authors to rewrite both. Especially because “reporting guidelines” are about content too. They point out the content elements that should not be absent from papers, so using the word “content” in the manuscript text is not really appropriate. Please, re-evaluate.We completely agree with the point you raise. We have re-written this point in the Abstract, Results and Conclusions. “Participants believed that checking adherence to guidelines goes beyond the role of peer reviewers and were concerned that the quality of peer review could be compromised. Reviewers are generally not expected to focus on reporting issues but on providing an expert view on the importance, novelty, and relevance of the manuscript.” 8) As I said above, the abstract is good, and there is room for growth (you have now 232 words). However, I could not find the conclusions in the abstract helpful. Could you please inform better what you did conclude? What “points raised in the survey” should be considered?We have re-written and expanded the Conclusions in the abstract. The abstract is now 295 words. 9) Checklists as forms. ...“assessing completeness of reporting is a subjective task”. This is one of the most important sentences in this manuscript, it tells us about using reporting guidelines checklists as evaluation tools — for what they have not been created. I saw no discussion about this in your paper.We have now discussed this point in the paragraph before the limitations of the study. 10) Discussion. The discussion of the paper is short, and it lacks several points pointed above. Mainly, it does not discuss the limitations and potential bias of this study. Discussing limitations is a requirement from F1000Research journal.We have now included such a section in the Discussion section. 11) You mention you did analyse qualitative information using NVivo. How did you do it?We have now provided further information on how we did the qualitative analysis with NVivo (“Data analysis” section). 12) There are at least eight items from the CHERRIES checklist that I could not see in your paper, and you might want to report, even if in a separate (or supplementary) box, from data protection, preventing multiple entries, to randomisation of items, and others.We have now included the information corresponding to the missing items throughout the whole Methods section. 13) From Box 1, I do not quite get the difference between Interventions 1 and 2. It seems that both ask authors to submit a populated/completed RG checklist. Is it that #1 asks for page numbers and #2 does not?#1 asks for page numbers and #2 for text from the manuscript. Some people may think that if you nudge authors to have to copy-paste text into the checklist they may tend to take it more seriously than if you just ask them to put a page number. We have slightly modified their wording in Box 1 to make it clearer. 14) In my opinion, the sentences from “To contact editors...” to “improve adherence to RGs” belong more to the “Participants” section than to the “Procedure”. Especially the first sentence. Also “Participants could suggest further editors that they considered could contribute to the survey” is something that I would consider as a snowballing or recruitment method, not the survey procedure per se. So, I would suggest putting everything related to recruitment together in one section, to make it easier for your reader (who might be interested in undertaking a similar survey) to find information on such a critical issue as recruitment. Also: how could participants suggest? Was it a specific question in the SurveyMonkey form?We have restructured that part of the manuscript and divided it into “Recruitment” and “Survey administration”. We have also provided the information requested. 15) Were participants given an estimation of the time required to answer the survey before they began?We have now included this information (“Survey administration” subsection). Minor: about citations16) Reference number 1 (EQUATOR Network website) is not a proper, complete reference. It points to the home page of the website, not to a document, and as so, it does not show from where the authors collected that information (the reader cannot find them in the home page). The correct way of citing would be adding the full URL. A suggestion: EQUATOR Network. What is a reporting guideline? Available at:http://www.equator-network.org/about-us/what-is-a-reporting-guideline/. Cited on 19/11/2019.Done. 17) Something similar happens to reference number 13. Author and title are missing. It would help readers if it could be typed like this:Schoter S. Evidence-based publishing. BMJ. Available at: https://www.bmj.com/about-bmj/evidence-based-publishing. Cited on 19/11/2019.Done. 18) Reference 14 also lacks authors and titles. References 13, 14, 16, 21 and 22 lack a lot of elements necessary for readers to be able to find them. When working in a printed version (as I am now), you cannot see what the document is, where it is published or by whom — these are available only by those reading online and able to “click” on the links.Done. 19) I would change the sentence:“Raw survey results are given as Underlying data20.”To something clearer and that does not require the reader to go to the reference list to understand what it is.“Table XX in the supplementary file X20 shows the responses from all 24 participants.”Done. 20) Has the study by Hawwash et al. (reference 25) been published yet? (The authors cited the protocol only). Do the authors plan to cite other electronic tools that are out there?We have now cited the results paper by Hawwash et al., which got published on 6 November. Regarding the electronic tools, we have already cited COBWEB and Statreviewer. We did not cite Penelope as it does not explicitly check RG requirements – it just helps authors submit the relevant RG checklist." } ] } ]	1	https://f1000research.com/articles/8-1682
https://f1000research.com/articles/8-2135/v1	20 Dec 19	{ "type": "Case Report", "title": "Case Report: Peripartum cardiomyopathy in a young female complicated by cardioembolic stroke", "authors": [ "Ravi Ranjan Pradhan", "Ajay Kumar Yadav", "Sant Yadav", "Prashant Kumar Gupta", "Ajay Kumar Yadav", "Sant Yadav", "Prashant Kumar Gupta" ], "abstract": "Stroke in a young female is rare but, when it occurs, has a serious economic, and mental burden to the patient, and family members. Peripartum cardiomyopathy (PPCM) is one of the rare causes of stroke in young females. We report a case of a 20-year-old female with PPCM complicated by cardioembolic stroke. The patient was started on long-term anticoagulation, and her heart failure regimen was optimized upon discharge. Anticoagulation therapy in cardioembolic stroke prevents further complications.", "keywords": [ "peripartum cardiomyopathy", "female", "stroke", "anticoagulation" ], "content": "Introduction\n\nPeripartum cardiomyopathy (PPCM) is a rare cause of heart failure affecting women in late pregnancy or in the puerperium. The overall incidence of PPCM ranges from 1 in 1300 to 1 in 15,000 pregnancies1. However, the incidence fluctuates globally, and is higher in developing countries2. PPCM is often recognized when the patient has severe myocardial dysfunction, less severe forms of PPCM often go unrecognized3. The 2010 European Society of Cardiology (ESC) Working Group defined PPCM as an idiopathic cardiomyopathy with following characteristics3:\n\n1. The development of heart failure (HF) towards the end of pregnancy or within five months following delivery.\n\n2. The absence of an identifiable cause of HF.\n\n3. Left ventricular (LV) systolic dysfunction with an LV ejection fraction (LVEF) of less than 45 percent. The LV may or may not be dilated.\n\nDespite many attempts to establish the exact etiology of PPCM, the cause remains unknown. Viral, autoimmune, dietary deficiencies, and idiopathic causes may contribute1. Familial occurrence of PPCM suggests a possible role of genetic predisposition1. Altered prolactin processing, and elevated soluble Fms-like tyrosine kinase 1 (Flt 1) have also been associated with the pathogenesis of PPCM4,5. During pregnancy, increased oxidative stress leads to cleavage of prolactin by cathepsin D into abnormal 16 kDa protein. This protein damages the heart, and blood vessels4. Soluble Flt 1 is secreted by placenta, that inhibits vascular endothelial growth factor signaling, which leads to angiogenic imbalance, and endothelial dysfunction5. Relaxin-2, a hormone produced by ovaries, breast, and placenta has a potential beneficial effect in PPCM. It increases cardiac output, and decreases vascular resistance6. Risk factors of PPCM are increased maternal age, increased parity, multiple pregnancy, malnutrition, use of tocolytics, and preeclampsia or eclampsia1. Patients usually present with shortness of breath, orthopnea, cough, hemoptysis, paroxysmal nocturnal dyspnea, and ankle edema3. Tachycardia, elevated jugular venous pressure, third heart sound (S3), and displaced apex beat are common, however; basal crackles are less common3. A high index of suspicion is required for diagnosis, as these features are common in advanced pregnancy and the peripartum period3.\n\nAbout 6 % of patients of PPCM present with thromboembolic complications such as deep vein thrombosis, pulmonary thromboembolism, stroke, acute limb ischemia etc7. Morbidity and mortality is high in PPCM, especially when associated with cardioembolic phenomenon3. Fortunately, despite the high morbidity, mortality, and a high risk of relapse in succeeding pregnancies, many patients with peripartum cardiomyopathy recover within three to six months of disease onset2. Here, we report a rare case of a young female with peripartum cardiomyopathy complicated by cardioembolic stroke.\n\n\nCase report\n\nA 20-year-old female farmer from Nepal presented with shortness of breath and cough which had lasted 10 days in November, 2019. The shortness of breath had a gradual onset and was progressive. Initially patient had shortness of breath on exertion only, but with time she had shortness of breath even at rest, and it was associated with orthopnea. The patients cough was productive with mucoid sputum lasting for 10 days. There was no history of fever, chest pain, and hemoptysis. The patient was 45 days post-partum, and was breastfeeding. She didn’t indicate history of illicit drug abuse and use of hormonal contraceptives. The family history was negative for premature coronary artery disease, young stroke, and premature death. With the above symptoms the patient present at her local hospital, where she received injectable and oral antibiotics (intravenous ceftriaxone 1 gram twice a day and oral azithromycin 500 mg daily) thinking that it could be a chest infection. However, her symptoms were not relieved and, she visited our center.\n\nOn examination the patient had a temperature of 36.7°C (normal = 36.5°C to 37.5°C), heart rate of 109 beats per minute (normal = 60 to 100 beats per minute), blood pressure was 100/60 mm Hg (< 120/80 mm Hg), respiratory rate of 20 breaths per minute (normal = 14 to 16 breaths per minute), and oxygen saturation of 92% (normal = 95% to 100%)while he was breathing ambient air. There was no peripheral edema. On chest examination there was dullness in the right infra-scapular and infra-axillary region and the intensity of breath sound was decreased, however her trachea was in the midline. Examinations of other systems were unremarkable.\n\nOn investigation total leukocyte count was 8,300/uL, hemoglobin was 11.4 gm/dL, platelet count was 4,35,00/uL, and random blood sugar was 100 mg/dL. Details are shown in Table 1.\n\nA provisional diagnosis of right sided pleural effusion was made and the patient was admitted for further evaluation. Antibiotic treatment (intravenous ceftriaxone 1 gram twice a day) was initiated. One day after hospital admission the patient developed weakness of left half of the body. The weakness was more in the upper limb compared to the lower limb. It was associated with slurring of speech, and deviation of face towards the right side. On neurological examination, the patient had left-sided upper motor neuron type facial nerve palsy, muscle strengths in the left upper and lower limbs were 0/5 and 1/5 respectively on the Medical Research Council (MRC) scale, and there was an ipsilateral Babinski sign. Fundoscopy findings were unremarkable. A computed tomography scan of the head was done immediately which was normal. A repeat CT scan head at 48 hours showed a wedged shaped hypodensity involving the gray and white matter of the right anterior temporal lobe, a hypodense area in the posterior portion of lentiform nucleus, the adjacent internal capsule region, and in the head of caudate nucleus (features suggestive of right sided acute ischemic stroke) [Figure 1]. Electrocardiogram showed sinus tachycardia, and poor progression of the R-wave [Figure 2].\n\nTransthoracic echocardiography (TTE) showed global hypokinesia of the left ventricular wall with an LVEF of 25%, moderate mitral regurgitation, mild tricuspid regurgitation, and minimal pericardial effusion. On transesophageal echocardiography (TEE) there was no left atrial or ventricle clot.\n\nA diagnosis of right sided ischemic stroke (cardioembolic) with peripartum cardiomyopathy was formulated. The patient was treated with aspirin 150 mg daily, frusemide 20 mg twice a day, spironolactone 25 mg daily, metoprolol 25 mg daily, and prophylactic unfractionated heparin (UFH) 2500 units subcutaneously twice a day. Limb physiotherapy was initiated. Two weeks after the incident of stroke, anticoagulation with warfarin 5 mg daily bridged with low molecular weight heparin 40 mg subcutaneously twice a day for an initial five days was administered. Aspirin and UFH were stopped. On discharge, her HF medications were optimized (frusemide 20 mg twice a day, spironolactone 50 mg daily, and metoprolol 50 mg daily), and anticoagulation with warfarin 5 mg daily was continued with provision for regular monitoring of prothrombin time (PT), and international normalization ratio (INR). At the time of discharge, her power was 3/5 and 4/5 in the left upper limb, and lower limb respectively. The patient was counselled about avoiding subsequent pregnancies.\n\n\nDiscussion\n\nStrokes in young adults are uncommon, comprising 10–15% of all stroke patients8,9. Though uncommon, stroke in young adults has a large economic burden, often leaving the victims disabled during their most productive years.\n\nPreviously published articles have defined the cut-off age for strokes in young adults as those younger than 45 or 49 years10. The etiologies and nature of strokes in young adults are different from the older patients. In young adults, congenital and acquired heart diseases, hematological conditions (such as sickle cell disease), vasculopathy (such as arterial dissection and vasculitis), pregnancy (cortical venous sinus thrombosis, preeclampsia/eclampsia), other hypercoagulable states, smoking, illicit drugs, premature atherosclerosis, hypertension, metabolic disorders (such as Fabry disease, homocystinuria etc), and possibly migraine are common causes The etiology of stroke in young patients remains a diagnostic challenge. In our patient, the etiology was cardioembolic secondary to hypokinesia of the left ventricle (EF=25%) due to peripartum cardiomyopathy. In a case report from Kumbham et al.11 the patient had multifocal infarct involving the left frontoparietal lobe, both occipital lobes, and the right insular cortex as well as thrombi in the left ventricle, which is in contrast with our case. In our case, we performed TEE to rule out a thrombus in the left atria or ventricle, the results of which were negative. The case report from Kumbham et al.11 had a multifocal infarct involving both hemispheres, but in our case the infarct was unilateral. Maternal age > 30 years is one of the risk factors for PPCM12, however our patient was 20 years old. Other conventional risk factors for PPCM such as increased parity, multiple pregnancy, use of tocolytics, and preeclampsia or eclampsia were not present in our patient.\n\nManagement of PPCM with cardio-embolic stroke requires a multidisciplinary approach that involves cardiologist, neurologist, obstetrics, and physiotherapist. Components of HF therapy in PPMC are similar to that of other types of HF, with attention to avoiding particular medications that have an effect on the fetus2. Angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs), angiotensin receptor-neprilysin inhibitors (ARNI), and aldosterone antagonists are teratogenic, and shouldn’t be used during pregnancy. In our patient we have avoided all these medications, but we used aldosterone antagonist (spironolactone) as our patient was 45 days post-partum, and is relatively safe during lactation13. Anticoagulation in PPMC is administered when LVEF is < 30 %14. In our patient (LVEF=25 %), anticoagulation was started at two weeks following the ischemic stroke to avoid the risk of bleeding, as the infarct was involving more than one-third of the middle cerebral artery region.\n\nThe risk of recurrent HF in patients with PPCM is around 25 % when LV function has recovered, and 50 % when LV function has not recovered15. We therefore, strongly counselled our patient to avoid subsequent pregnancies.\n\nLimitations: Patient developed acute ischemic stroke during hospital stay, however; we were not able to thrombolyse the patient with intravenous recombinant tissue plasminogen activator (rtPA) therapy due to financial constraints for the patient. Work-up for thrombophilias, and vasculitis disorders were not feasible for the same reason. At presentation to our center, we missed the diagnosis of PPCM initially, and didn’t consider that pleural effusion could be due to HF in PPCM.\n\n\nConclusion\n\nPPCM is one of the rare causes of HF in puerperium. One should consider PPCM as a differential diagnosis in any patient presenting with shortness of breath, and cough during puerperium. Stroke in PPCM is rare, but has a devastating effect on mother, infant, and other family members. Early diagnosis, optimization of HF medication, and anticoagulation therapy prevents the further complications. Patient counselling to avoid subsequent pregnancies is the cornerstone to prevent recurrent HF.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required", "appendix": "References\n\nAbboud J, Murad Y, Chen-Scarabelli C, et al.: Peripartum cardiomyopathy: A comprehensive review. Int J Cardiol. 2007; 118(3): 295–303. PubMed Abstract \| Publisher Full Text\n\nBhattacharyya A, Basra SS, Sen P, et al.: Peripartum cardiomyopathy: a review. Tex Heart Inst J. 2012; 39(1): 8–16. PubMed Abstract \| Free Full Text\n\nSliwa K, Hilfiker-Kleiner D, Petrie MC, et al.: Current state of knowledge on aetiology, diagnosis, management, and therapy of peripartum cardiomyopathy: a position statement from the Heart Failure Association of the European Society of Cardiology Working Group on peripartum cardiomyopathy. Eur J Heart Fail. 2010; 12(8): 767–78. PubMed Abstract \| Publisher Full Text\n\nForster O, Hilfiker-Kleiner D, Ansari AA, et al.: Reversal of IFN-gamma, oxLDL and prolactin serum levels correlate with clinical improvement in patients with peripartum cardiomyopathy. Eur J Heart Fail. 2008; 10(9): 861–8. PubMed Abstract \| Publisher Full Text\n\nPatten IS, Rana S, Shahul S, et al.: Cardiac angiogenic imbalance leads to peripartum cardiomyopathy. Nature. 2012; 485(7398): 333. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDamp J, Givertz MM, Semigran M, et al.: Relaxin-2 and soluble Flt1 levels in peripartum cardiomyopathy: results of the multicenter IPAC study. JACC Heart Fail. 2016; 4(5): 380–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWong CM, Hawkins NM, Jhund PS, et al.: Clinical characteristics and outcomes of young and very young adults with heart failure: The CHARM programme (Candesartan in Heart Failure Assessment of Reduction in Mortality and Morbidity). J Am Coll Cardiol. 2013; 62(20): 1845–54. PubMed Abstract \| Publisher Full Text\n\nVarona JF, Guerra JM, Bermejo F, et al.: Causes of ischemic stroke in young adults, and evolution of the etiological diagnosis over the long term. Eur Neurol. 2007; 57(4): 212–8. PubMed Abstract \| Publisher Full Text\n\nGeorge MG, Tong X, Kuklina EV, et al.: Trends in stroke hospitalizations and associated risk factors among children and young adults, 1995-2008. Ann Neurol. 2011; 70(5): 713–21. PubMed Abstract \| Publisher Full Text\n\nSmajlović D: Strokes in young adults: epidemiology and prevention. Vasc Health Risk Manag. 2015; 11: 157–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumbham P, Sharma B, Patel A, et al.: POSTPARTUM CARDIOMYOPATHY WITH CARDIOEMBOLIC STROKE (P3.298). Neurology. 2017; 88(16 Supplement): P3.298. Reference Source\n\nWhitehead SJ, Berg CJ, Chang J: Pregnancy-related mortality due to cardiomyopathy: United States, 1991-1997. Obstet Gynecol. 2003; 102(6): 1326–31. PubMed Abstract \| Publisher Full Text\n\nPhelps DL, Karim A: Spironolactone: relationship between concentrations of dethioacetylated metabolite in human serum and milk. J Pharm Sci. 1977; 66(8): 1203. PubMed Abstract \| Publisher Full Text\n\nHowie PW: Anticoagulants in pregnancy. Clin Obstet Gynaecol. 1986; 13(2): 349–63. PubMed Abstract\n\nElkayam U, Goland S, Pieper PG, et al.: High-risk cardiac disease in pregnancy: part II. J Am Coll Cardiol. 2016; 68(5): 502–16. PubMed Abstract \| Publisher Full Text" }	[ { "id": "66969", "date": "30 Jul 2020", "name": "Guillaume Schurtz", "expertise": [ "Reviewer Expertise acute heart failure", "cardiogenic shock", "mechanical circulatory support", "percutaneous coronary interventions", "TAVR" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this case, authors aimed to discuss the epidemiology of cardioembolic stroke in young adults (i.e. females) and illustrate this with a possible diagnosis of PPCM. Manuscrpit is well written but many questions remained. PPCM diagnosis is very difficult and cannot be confirmed in this case. Indeed, in the absence of TTE evaluation before delivery (or in the month before) demonstrating usually parameters observed in pregnant women without other abnormalities (LVEF dysfunction,...), we cannot distinguish a PPCM from another form of previous unknown dilated cardiomyopathy. This is a major pitfall in this manuscript, but also seriously raised concern about the true existence of PPCM diagnosis. Of note, LV diameters are not available, nor aortic valvular evaluation or arguments (or not) for a eventual congenital cardiac disease. Cardiac biomarkers are absent, and extensive coagulation testing must have been realized to exclude a possible diagnosis of catastrophic thrombotic syndrome (aPLS, TTP...). Moreover, despite the absence of robust evidence with its utilization (and also the thrombotic risk), bromocriptine prescription should however have been discussed.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] }, { "id": "90531", "date": "06 Aug 2021", "name": "Prashant Nasa", "expertise": [ "Reviewer Expertise Critical care medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI congratulate the authors for the successful diagnosis of this case despite resource limitations. I have few suggestions for discussion to make it helpful for readers.\nDiagnosis of peripartum cardiomyopathy should be explained as the patient has no previous history of cardiac dysfunction (recent uneventful delivery, with normal cardiac valvular status). The diagnosis of peripartum cardiomyopathy is an exclusion diagnosis.\n\nAnticoagulation management (therapeutic and secondary prophylaxis) needs to be discussed. There are no standard recommendations for anticoagulation management for stroke in peripartum cardiomyopathy. A recent case report, (Nasa et al., 2021), discussed this controversy. The dilemma of delaying anticoagulation (for two weeks) to avoid hemorrhagic transformation of infarct area vs early anticoagulation in a prothrombotic state of peripartum cardiomyopathy. This is important for secondary prophylaxis.\n\nThrombophilic profile is required to exclude the differential diagnosis of thrombophilia.\n\nDuration of follow-up and anticoagulation is also required. This is missing, as many cases are reversible so long term anticoagulation is not required, as noted in this case report.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] } ]	1	https://f1000research.com/articles/8-2135
https://f1000research.com/articles/8-2123/v1	19 Dec 19	{ "type": "Opinion Article", "title": "Re-examining physician-scientist training through the prism of the discovery-invention cycle", "authors": [ "Gopal P. Sarma", "Allan I. Levey", "Victor Faundez", "Allan I. Levey", "Victor Faundez" ], "abstract": "The training of physician-scientists lies at the heart of future medical research. In this commentary, we apply Narayanamurti and Odumosu’s framework of the “discovery-invention cycle” to analyze the structure and outcomes of the integrated MD/PhD program. We argue that the linear model of “bench-to-bedside” research, which is also reflected in the present training of MD/PhDs, merits continual re-evaluation to capitalize on the richness of opportunities arising in clinical medicine. In addition to measuring objective career outcomes, as existing research has done, we suggest that detailed characterization of researchers’ efforts using both qualitative and quantitative techniques is necessary to understand if dual-degree training is being utilized. As an example, we propose that the application of machine learning and data science to corpora of biomedical literature and anonymized clinical data might allow us to see if there are objective “signatures” of research uniquely enabled by MD/PhD training. We close by proposing several hypotheses for shaping physician-scientist training, the relative merits of which could be assessed using the techniques proposed above. Our overarching message is the importance of deeply understanding individual career trajectories as well as characterizing organizational details and cultural nuances to drive new policy which shapes the future of the physician-scientist workforce.", "keywords": [ "metascience", "translational research", "biomedical policy", "innovation", "medical education", "data science", "MD/PhD", "bench-to-bedside", "physician-scientist", "clinical research" ], "content": "Introduction\n\nThe origin of the modern physician-scientist lies in the explosive growth of knowledge following the Second World War. Although a recognizable vision for the dual-degree training of clinical investigators was articulated as early as the second decade of the 20th century, it was the launching of the National Institutes of Health (NIH)-backed Medical Scientist Training Program (MSTP) in 1964 in the US that cemented the MD/PhD into the framework of modern medical research (Harding et al., 2017; Meltzer, 1909).\n\nIn the half-century since then, growth of the physician-scientist pipeline in the United States has expanded to nearly 120 schools, with over a third funded through the NIH (Akabas et al., 2018; Jeffe et al., 2014). Paralleling this growth has been a steady stream of meta-scientific research aimed at analyzing the career trajectories of dual degree graduates. With titles such as “The MD/PhD Researcher: What Species of Investigator,” “Transforming Science Into Medicine: How Clinician–Scientists Can Build Bridges Across Research’s ‘Valley of Death,’” and “Challenges and opportunities for reinvigorating the physician-scientist pipeline,” the fundamental aim of this type of analysis is to use systematic, evidence-based investigation into the frontiers of medical research to inform actionable policy (Andreassen & Christensen, 2018; Daye et al., 2015; DeLuca et al., 2016; O’Mara et al., 2015; Roberts et al., 2012; Rosenberg, 2008; Sklar, 2017; Sutton & Killian, 1996; Thiele, 2018; Weggemans et al., 2019).\n\nIn this commentary, we contribute to this knowledge-base by applying the framework of “discovery-invention cycles,” as proposed by Venkatesh Narayanamurti and Toluwalogo Odumosu, to the training of physician-scientists (Narayanamurti & Odumosu, 2016). Their framework can be seen both as a theory of scientific progress as well as a theory of innovation—we will draw on both perspectives in this article. We begin by summarizing their argument for the inadequacy of the linear model of research and for dismantling the artificial distinctions between “basic” and “applied” research, which we equate with notions of “bench” and “bedside” research in the medical setting. We argue that the “bench-to-bedside” paradigm of clinical-translational research is a manifestation of the linear model, which informs the structure of the integrated MD/PhD program. We propose that re-framing clinical-translational research in the language of discovery-invention cycles allows us to better align the training of physician-scientists with 21st-century needs and opportunities.\n\nNext, we examine the obstacles to accurately measuring outcomes in dual-degree programs. We argue that objective measures related to job description, such as employment at an academic center, percentage of time spent conducting research, and field of research, give a limited window into whether the unique training of dual-degree graduates is being utilized. As a complementary research paradigm, we propose that more in depth, anthropological research is necessary to understand the specific ways in which individuals are or are not taking advantage of their clinical and scientific background. As a set of tools to aid qualitative investigation, we propose that machine learning and data science of large biomedical corpora along with anonymized clinical data may allow us to better understand if there are objective “signatures” of MD/PhD training.\n\nFinally, we examine several pathways for re-thinking the training of physician-scientists: shifting the start of the PhD portion of MD/PhD programs to after the required clinical rotations; expanding the pipeline of residency/fellowship-based PhD programs; recruiting PhDs, other advanced degree holders, and professionals from relevant disciplines into the medical pipeline; and elevating the MD as a terminal clinical degree for a subset of individuals interested in non-clinical careers. Although most of these strategies are employed to varying degrees in existing programs, there is little objective basis on which to decide the relative proportion of each type of training in a national funding roadmap. Our proposals for collecting richer data will play a critical role in allowing policy makers in the biomedical sciences to better understand the consequences of different training paradigms. Taken together, the framing of the discovery-invention cycle, anthropological investigation, and the tools of modern data science will allow the medical establishment to navigate rapidly evolving intellectual frontiers at the interface of medicine and other disciplines.\n\nAlthough much of our discussion is US-centric, our analysis is relevant for other countries as well, as the American model has largely been replicated elsewhere. Moreover, dual-degree physician-scientist training appears to play a significantly smaller role in the landscape of medical research outside of the US (Alamri, 2016). Consider, for example, that in the UK, the first integrated MB/PhD program was introduced at the University of Cambridge in 1989. In Australia, the first combined MBBS1/PhD was introduced at the University of Sydney in 1998, but was subsequently scaled back significantly in 2014. In Africa, the University of Cape Town recently created a program in which students apply for additional research training in a stepwise fashion extending from a bachelors in medicine, to a masters, and finally to a PhD. This cohort began its training in 2011 and career outcomes are not yet available (Alamri, 2016; Kandiah, 2013; Katz et al., 2014). For countries and regions with fledgling efforts in physician-scientist training, we believe that our analysis will prove to be useful in establishing a vision and long-term roadmap for creating sustained innovation in the biomedical workforce.\n\n\nThe discovery-invention cycle\n\nIt is apparent then that the natural sciences and engineering have long been interlinked and that practical pursuits have never been far from the acquisition of new knowledge. However, in contemporary US science and technology policy and in the governance of many of our mission-oriented research agencies, this simple idea has been forgotten and silos and boundaries have built-up, impeding the progress of knowledge and inventions. The status quo is unsustainable and unacceptable.\n\n- Venkatesh Narayanamurti and Toluwalogo Odumosu in Cycles of Invention and Discovery: Rethinking the Endless Frontier (Narayanamurti & Odumosu, 2016)\n\nNarayanamurti and Odumosu’s basic tenet is a simple one. They observe that advances that are traditionally considered the domain of “fundamental” research are often richly intertwined with more “applied” or “development” related work. This observation has deep, practical implications for science innovation, training, and policy. Because of the assumption that fundamental research has a linear relationship with applied research, policymakers at universities, funding agencies, or industrial research labs assume that increasing the pipeline of fundamental research will necessarily increase practical output at the other end. However, the reality is far more complex. The road from fundamental insights to practical technologies or other applications is strewn with many more obstacles and opportunities than this simple picture allows for. Consequently, policies aimed at increasing innovation by attempting to engineer aspects of this linear pipeline may be less than successful. In particular, thinking in terms of the linear model can result in institutions that fail to adequately identify and/or resolve bottlenecks to science innovation and training.\n\nWe will return to the question of physician-scientist training in the subsequent section and argue that the bench-to-bedside paradigm is a manifestation of a linear model in need of revision. Before turning our attention to medicine, however, we first examine 12 Nobel Prizes in telecommunications technology and nuclear magnetic resonance (NMR) that Narayanamurti and Odumosu use to illustrate the richly interdependent and non-linear nature of basic and applied research (Narayanamurti & Odumosu, 2016). There are several points worth understanding in taking a bird’s eye view of this Nobel Prize winning work.\n\nThe first is the complex interplay between advances firmly on the discovery side and those on the invention side. Most illustrative is the 1956 Nobel Prize in Physics, awarded to John Bardeen, William Shockley, and Walter Brittain for the discovery of the transistor effect (Riordan et al., 1999). In Figure 1, this award is firmly situated at the boundary of the discovery and invention territories. The reason is that not only did the transistor effect represent a fundamental advance in understanding interactions between electromagnetic fields and metals, but in addition, demonstrating the effect required building the bipolar-contact transistor. Indeed, eminent theorist John Bardeen spent countless hours alongside his experimental colleagues at Bell Laboratories during the development of this groundbreaking work. Likewise, the development of nuclear magnetic resonance (NMR), a discovery which ultimately came to transform medicine, required parallel insights into engineering technology, which would in turn allow further theoretical developments in quantum physics and quantum chemistry (Boesch, 2004; Tabet & Rebuffat, 2003). The development of the molecular ray method and the discovery of the proton magnetic moment played an analogous role to NMR as the transistor did for entire branches of solid state physics, and consequently, are situated firmly on the boundary between discovery and invention (Stern, 1946; see Narayanamurti & Odumosu, 2016 for an analogous diagram of interconnected Nobel Prizes leading to NMR).\n\nArrows indicate conceptual dependencies among the various inventions and discoveries. Figure has been adapted with permission from (Narayanamurti et al., 2013).\n\nThe second point that Narayanamurti and Odumosu conclude from these historical developments is that building an institutional culture that allows such interdependent connections to be formed requires administrators to maintain both an overarching thematic focus along with a flexible and open-minded view towards the necessary diversity of prior training and experiences on a team. As the above examples are meant to illustrate, the series of steps that were required for this ground-breaking research to be conducted often took a meandering path spanning theoretical work, laboratory experiments, and practical industrial development. A rigid environment that prioritized either research publications, patents, or commercial developments to the exclusion of the others would have failed to deliver on the true potential of this work.\n\nWe summarize the basic takeaways from the discovery-invention cycle paradigm here:\n\n1. All aspects of knowledge production should be intrinsically valued. Realizing the social impact of any advance requires many intricate steps, which can intertwine theoretical research, fundamental science, technological or product development, as well as managerial and organizational insights. A key element is the convergence of diverse intellectual assets.\n\n2. The distinction between basic and applied research (or bench and bedside, in the case of medicine)—and the greater prestige that modern society affords either basic or applied research, depending on the context—can be detrimental to efficient resource allocation across the intellectual landscape.\n\n3. Creating an institutional culture that maximizes the potential for innovative impact requires breaking down artificial boundaries and enabling dialogue not just across disciplines, but across the different dimensions of the discovery-invention cycle.\n\n\nMeasuring the impact of dual-degree training\n\nDespite the remarkable success of the biotechnology sector and the embrace of its approaches by the pharmaceutical industry, a functioning LSM [life sciences and medicine] continuum linking fundamental discovery and the care of patients remains, for the most part, a goal rather than a reality.\n\n- ARISE2 Committee (American Academy of Arts and Sciences, 2013)\n\nThe discovery-invention cycle can be considered both a theory of scientific progress as well as a theory of innovation. In applying this framework to medicine, we take the latter perspective and consider that at its core, innovation consists of novel amalgams of existing concepts, skills, methodologies, and resources, which in turn give rise to fundamentally new ones (Baregheh et al., 2009; Fagerberg et al., 2005). Theoretically, the greater the number, diversity, and depth of the clinical and scientific training of individuals, the greater the possibilities for novel combinations of concepts, skills, methodologies, and resources. We see the original vision of the MD/PhD as being in this spirit—to increase innovative outcomes at the interface between basic science and clinical research.\n\nPhysician-scientists are meant to play the unique role of bridge builders in the vast landscape of the basic and clinical sciences (American Academy of Arts and Sciences, 2013; Zemlo et al., 2000). What is the nature of these bridges and how should we train future leaders to help construct these key edifices? The notions of “bench to bedside” and “bedside to bench” hide the complex realities which underlie both of these research and development pathways. A ‘pure’ example of formulating testable hypotheses from clinical observations is John Snow’s discovery of the causes of cholera outbreaks in 19th century London, even before the advent of the germ theory (Johnson, 2006). Likewise, it is expected that physician-scientists enable the subsequent steps of translational research, which will give rise to novel therapies from bench research.\n\nUnfortunately, both sides of this process invariably involve complex detours that hamper the attempts of the well-trained physician-scientist. One example of the tortuous nature of both the “bench to bedside” and “bedside to bench” trajectories is manifest in psychiatric genetics. Schizophrenia and mood disorders are characteristically polygenic, thus preventing the formulation of biological pathogenesis hypotheses from classical Mendelian genetic approaches (International Schizophrenia Consortium et al., 2009). It was the advent of powerful chemical DNA synthesis tools, DNA hybridization on chips, DNA sequencing technologies, robotics, novel statistical tools, and the computer power to analyze and store genomic and clinical data from thousands of patients and controls that has opened the door to formulate biological hypotheses of the underlying disease mechanisms (Geschwind & Flint, 2015; Sullivan et al., 2012). While it is not our aim to undertake an analysis as thorough as Narayanamurti and Odumosu, there is no shortage of clinical breakthroughs whose trajectory was as meandering and boundary crossing as the advances in solid state physics and NMR summarized above. Consider the example of statins, drugs that are widely used to treat hypercholesterolemia. Their discovery and clinical applicability needed the convergence of knowledge about natural product chemistry, cholesterol metabolism, and the genetics of hypercholesterolemia disorders, to mention just a few (Stossel, 2008). In both of these cases, schizophrenia and statins, fundamental science, technological / product development, and managerial / organizational insights have been key enablers of research progress.\n\nOn the one hand, many will point out that these observations are hardly surprising. To some degree, the diversity of efforts required to actualize a given medical therapy—basic or applied, academic or industrial—is a well understood phenomenon. On the other hand, if so, is this reality reflected in how we train our physician-scientists? And what of the expected career trajectories of dual-degree graduates? Does the ideal physician-scientist career path optimally take advantage of the unique nature of their training? What policy changes can be implemented to fully realize the intellectual potential of our medical establishment and our arsenal of dual-degree graduates?\n\nAs we discussed in the introduction, there is a sizeable body of metascientific research analyzing the impact of MD/PhD training. Most recently, in the United States, the Association of American Medical Colleges conducted a thorough and thoughtful investigation into nearly 50 years’ worth of career outcomes of dual-degree graduates (Akabas et al., 2018). To assess the institution-wide impact, they examine a wide variety of measures including employment at an academic center, presence in major grant databases, and percentage of protected research time, among others. The AAMC study is an impressive first-pass analysis characterizing the landscape of dual-degree career outcomes. Given the complex, multi-faceted nature of physician-scientist training and practice, their report paves the way for subsequent investigations that can build on this foundation.\n\nOne of the conclusions of the AAMC study is that many questions remain in understanding the nature of the research landscape of physician-scientists. As an example, although the authors went into the study expecting a bimodal distribution of research time (i.e. with most being primarily clinicians or pure researchers), they discovered that there is in fact a continuum of research effort. Beyond a high-level description of their research activities, a major open question remains as to how researchers are taking advantage of their training apart from maintaining parallel job descriptions in varying proportions.\n\nOur primary takeaway from applying the lessons of the discovery-invention cycle to medical research is that there is a significant need for in-depth qualitative analysis to better characterize whether dual-degree training is truly being utilized. Even if we can establish beyond the shadow of a doubt that MD/PhDs are deeply involved in both clinical and research activities, there is still the lingering question of whether or not they are leveraging their unique and extensive training. If we were to eliminate the dual-degree trajectory entirely, would the corresponding research output vanish, or would it be accomplished in different ways by pure MD and PhD researchers? Or is it that the true contribution of MD/PhDs is to facilitate dialogue between researchers and clinicians through higher-level administrative and organizational activities? The AAMC study is an impressive cross-sectional examination of the entire cohort of MD/PhD trainees giving policymakers high-level structural insights and subsequent analyses can be more targeted and examine smaller groups of individuals in significantly greater depth. Examining the roles, activities, and intellectual philosophy of physician-scientists even from a single academic organization (school, department, or division) would give significant insight into the impact of the training.\n\nThe application of data science to public corpora of scholarly publishing is allowing researchers to investigate novel hypotheses about the scientific process itself. These new research trends are being embraced in the life sciences (see, for example, the Meta-Research collection in the journal eLife). We can now analyze complex datasets to study the evolution of scientific language since the 19th century or the frequency of medical reversals in the literature on clinical trials, and use the results to inform policy decisions (Herrera-Perez et al., 2019; Plavén-Sigray et al., 2017).\n\nLikewise, we argue that modern computational techniques may have a unique role in augmenting the qualitative, anthropological analyses we have argued for in this piece. Indeed, quantitative techniques have a long history in the study of innovation (Hall & Jaffe, 2012; National Academies of Sciences, Engineering, and Medicine, Division of Behavioral and Social Sciences and Education, and Committee on National Statistics, 2017). Imagine that we have a fully-digitized corpus of biomedical literature that can be used by data scientists. Such a resource would consist of aggregated articles from biomedical journals along with the tools for conducting large scale text searches, visualization, and the training of machine learning models. Is it possible that lying hidden in such datasets are signatures of research uniquely enabled by MD/PhD training?\n\nOne way to approach this question is from a purely exploratory standpoint. What are the patterns that emerge when looking at the research output of MD/PhD investigators? Are there differences in the distributions of journals that they publish in relative to pure MDs or PhDs? What about the distribution of topics? What about correlating trends in academic publishing from MD/PhDs with other sources, such as biomedical news or patent databases?\n\nAnother way to approach this question is the paradigm of classification (Bishop, 2006). Suppose for every research publication in our dataset, we extract the educational qualifications of the authors (since many medical journals require this, we might even restrict to those papers that list the qualifications of authors). We can then use supervised learning to create models that predict whether a dual-degree graduate co-authored the research study. To simplify the analysis, we could focus on the first author, senior author, or perhaps train a model to predict how many MD/PhD degree holders were involved with the study. How accurate might such a model be? If it turns out that no such model can be trained to perform better than chance, does that imply that the research output of MD/PhDs could just as well have come from pure MDs or PhDs? On the other hand, if it turns out that a model can be trained to achieve high classification accuracy, does it imply the converse? Or might there be some latent structural explanation subtly biasing the model? These are all tantalizing questions that would require deeper investigation and which would add significant depth and new dimensions to the types of questions identified above.\n\nEven more powerful would be to combine corpora of biomedical literature with anonymized medical records and even patent databases. For example, are there differences in documentation patterns between pure MDs versus MD/PhDs? What about in the standard of care? One would hope that there are no differences in routine cases, but what about in difficult situations where there is no standard of care? Are MD/PhDs able to leverage their knowledge and experience with the scientific literature to impact patient care? Analogous questions might be asked using combinations of the biomedical literature with patent databases to understand the role of MD/PhDs in the process of translating discovery to invention.\n\nWe make this proposal with some amount of trepidation and emphasize that the types of analysis proposed here need to be anchored to solid, anthropological investigation. Without a deep understanding of the context and culture of MD/PhD training, purely data-driven investigations might easily be misinterpreted and give credence to erroneous policy decisions.\n\n\nTraining proposals\n\nPhysician-scientists are trained to ask clinically relevant questions in a health research environment that lead to the development of research projects linking basic and clinical sciences. Physician-scientists are also a vital force in transforming clinical observations into testable research hypotheses and translating research findings into medical advances.\n\n- Tamara R. Zemlo, Howard H. Garrison, Nicola C. Partridge, and Timothy J. Ley in “The Physician-Scientist: Career Issues and Challenges at the Year 2000” (Zemlo et al., 2000)\n\nWe now turn to several proposals to align the training of physician-scientists with the insights of the discovery-invention cycle. As we have discussed in the previous section, understanding the impact of different training paradigms is itself a complex question with a many-year time horizon from intervention to effect. Therefore, we present these proposals partly as hypotheses or topics for discussion that merit greater investigation using some of the techniques identified above. Several of the proposals we discuss have been implemented to varying degrees at existing universities. However, with the integrated MD/PhD emerging as the dominant form of physician-scientist training, it remains an open question what the relative proportions should be of the different pathways. Again, the techniques proposed above may prove useful in answering this question.\n\nFor existing integrated MD/PhD programs, shifting the start of the PhD until after a full year of clinical rotations. For the traditional MD to residency track, expanding the pool of residency/fellowship-based PhD programs. In both cases, encouraging the PhD student to have both a true research mentor and a true physician-scientist mentor.\n\nOne of the most significant drawbacks of the linear model of research and development is a failure to adequately identify bottlenecks in the convoluted path from discovery to invention. Unfortunately, the current model of integrated MD/PhD training solidifies this model by sequentially training students in the pre-clinical sciences, followed by basic research, followed by clinical rotations. In the vast majority of cases in the US, students begin their PhD after having completed the pre-clinical years and taken Step 1 of the United States Medical Licensing Examination (USMLE), but before their required third-year clerkships.\n\nThis setup results in students choosing their PhD topic having had a limited exposure to the actual practice of clinical medicine. Consequently, students are not adequately prepared to identify a research path that is informed by experience with medicine itself. Shifting the start of PhD training to after the completion of third-year clerkships would give students substantially better preparation to choose a PhD area that would best serve their vision of being a physician-scientist in the context of an area of clinical scholarship that they wish to embark upon.\n\nAt some schools that have moved to an accelerated format for the pre-clinical years, in which Step 1 of the USMLE is taken after only 1–1.5 years of coursework, MD/PhD students are beginning their PhD after all or some of the clinical clerkships. See, for example, the MSTP program at Duke University, which has a one-year pre-clinical curriculum, or Baylor University, which has a 1.5-year pre-clinical curriculum. We endorse this path and our recommendation stands for schools that have maintained the traditional two-year format for pre-clinical training. For schools that have adopted a 1.5-year pre-clinical curriculum, but where MD/PhD students begin their graduate studies immediately after Step 1 of the USMLE, we recommend completing six months of clinical clerkships along with their traditional MD classmates prior to beginning their PhD, and perhaps also including 1–2 months of elective rotations in the areas relevant to the research they expect to pursue. An additional benefit of this arrangement is that students would be able to maintain their clinical skills during their PhDs. For example, an afternoon in an outpatient clinic once or twice a month would allow them to remain in contact with the clinical world while in the depths of their PhD research. It would also provide a valuable point of structure and regularity in the otherwise open-ended (and often soul searching!) years of graduate training.\n\nThe motivation behind expanding the pool of residency/fellowship-based PhD programs is similar in spirit. By residency/fellowship-based PhD programs, we are conceiving of a curriculum in which the majority of a student’s research is done during dedicated years separate from clinical training. We are skeptical that quality research can be conducted entirely in parallel with the grueling demands of residency and fellowship. See the Stanford ARTS Program or the UCLA STAR Program for outstanding models of research curricula integrated with post-graduate medical training. In both cases, the motivation is to ensure that students have greater exposure to clinical medicine before beginning their graduate careers. Moreover, residency/fellowship-based PhD programs open the door to many more students whose interest in a research career took a longer time to develop than those who decided early in their college careers apply to an integrated MD/PhD program.\n\nResidency/fellowship-based PhD programs also carry the benefit that students are more mature than had they began their PhDs soon after completing their undergraduate degree. Such students are likely to bring a more informed perspective to their graduate studies. Compared to typical first-year graduate students, they will already have substantial exposure to the scientific and clinical literature and will have been surrounded by research for many years, even if they have not yet been immersed in it. Moreover, because they are close to being fully-trained physicians, they can continue their clinical responsibilities during their PhD years in outpatient clinics or while moonlighting.\n\nFor residency/fellowship-based PhD programs, many will correctly point out that the elephant in the room is length of time it takes to train and the corresponding financial implications. Most residents interested in pursuing a PhD did not have the benefit of a subsidized MD/PhD program, and thus are saddled with significant loans from medical school. They are also at a time in life when they may have a new marriage, are planning a family, etc., with competing demands on time and other resources. It becomes very difficult for most individuals in this situation to turn down a starting job with a well-remunerated salary, and taking several more years to pursue the PhD. There will likely need to be innovative, but perhaps costly solutions to deal with these realities. However, we remain optimistic that such solutions are possible. Individuals frequently make career decisions that are far from optimal financially, but which give them a deep satisfaction in their life choices. Even a career in academic medicine, particularly in the setting of a dual-income household, is more than livable, and we suspect that a small, but sizeable student body would be enthusiastic to participate in such a set of programs.\n\nIn both integrated MD/PhD and residency/fellowship-based PhD settings, appropriate forms of mentorship would be necessary to have maximal impact. Specifically, it should be encouraged for students to have both a research mentor and a physician-scientist mentor.\n\nCreating a pipeline in which PhDs in other subjects or professionals of any kind with advanced training are recruited to the medical pipeline. For non-traditional MD candidates, ensuring a culture of mentorship and freedom that will allow them to develop their vision for integrating their prior experiences into a medical career.\n\nOne of the aims of viewing physician-scientist training from the lens of the discovery-invention cycle is to enable medical culture to be flexible in the face of a rapidly evolving landscape and emerging interfaces with other subjects. We believe that a sensible approach to creating such innovation and agility that would require minimal administrative overhead is to recruit PhDs as well as professionals from other disciplines to the standard medical pipeline. There is a long history of individuals who have made the decision to transition from a PhD to an MD. However, what we are advocating is a more ambitious and targeted recruiting culture within the medical establishment itself. The significant challenge, therefore, is how to discern those who are committed to a career as a physician-scientist, which is the minority, from those who have found they no longer see themselves pursuing a career in research and want a new direction in life.\n\nActively identifying and recruiting highly trained professionals (PhDs or otherwise) has the advantage that individuals bring a mature worldview to medical school and set of novel skills that can inform their clinical training and research focus. A small number of schools have created accelerated MD programs for biological science PhDs. See for example, the 3-Year PhD-to-MD-Program at Columbia University. Moreover, in situations where there is an immediate need for insights from a field outside of medicine, trained professionals can begin to make small contributions from the very beginning and are no more than four years away from becoming resident physicians. On the other hand, recruits to the standard MD/PhD pipeline are many years away from being able to contribute their knowledge base to medicine, even if their PhD topic has been strategically chosen to address a current need. Certainly, some creativity will be necessary to execute such a recruitment process, given the personality, skill set, and passion required for success in clinical medicine. However, considering that the demands and rewards of medicine are well understood by the educated public, and given the long history of individuals in medicine who have gone through significant career changes, we are confident that such a targeted recruitment process is achievable by sufficiently motivated medical school admissions committees.\n\nAs one contemporary example, we mention the intensely controversial discussions surrounding electronic health records (EHRs). Many believe that EHRs have negatively impacted the job satisfaction of healthcare workers as well as patient satisfaction. Indeed, research articles have specifically examined the role that EHRs play in increasing burnout among physicians (Arndt et al., 2017; DiAngi et al., 2016; Shanafelt et al., 2016). However, among the many voices in this discussion, we observe that one voice is notably absent: that of professional software engineers and user interface designers who could lend insights into why the situation in medicine has had such a disappointing outcome when we are surrounded by high-quality software in other arenas. Recruiting professional software engineers to the medical pipeline would allow these highly skilled individuals to contribute their insights to medical culture and establish the foundations for future advances at the intersection of software, medicine, and artificial intelligence.\n\nThe importance of mentorship in this process cannot be overstated. Unlike mathematics or theoretical physics, where one can be a prodigious scientist at a young age, the human and institutional complexity of medicine means that years of experience are typically required before an individual can speak with authority and generate mature insights. For those who are entering medical training after having established themselves in another field, being mentored by someone experienced in medicine can play a significant role in finding avenues to allow their existing knowledge base to become intertwined with medicine.\n\nThere are several exciting developments in science publishing that can be powerful enablers of this process. The use of “pre-prints” as part of the life cycle of idea generation, a practice that has been commonplace in some subjects for nearly 30 years, and which is now becoming more common practice in biomedicine, opens the door for experienced medical students, such as those with PhDs in other areas, to write scholarly articles on topics of medical interest but from a novel and integrated perspective that become part of the citable scientific literature. Likewise, the emergence of “open science,” and large-scale, distributed scientific collaborations may be particularly valuable ways for trained professionals to identify topics and projects of relevance to medicine that may not yet exist at their home institution (Nielsen, 2011; Sarma & Faundez, 2017).\n\nEncouraging a culture that values the MD as a terminal degree for individuals interested in non-clinical career paths such as medical data science, product development, consulting, science journalism, program management, venture capital or other positions that would advance the discovery-invention cycle across the life sciences.\n\nAn implicit assumption in analyses of the integrated MD/PhD program is that ideal outcomes involve individuals who maintain dual clinical and research responsibilities. However, this assumption rests on a linear view of bench-to-bedside research in which the role of the physician-scientist is to make testable hypotheses at the bench based on observations made at the bedside. If the reality of this pathway is far more complex, then perhaps it is not simply the specifics of training that should be reconsidered, but also the ideal career trajectory that individuals aspire to. Indeed, is it possible that success stories of dual-degree training may involve careers with neither research nor clinical responsibilities?\n\nThe dramatic growth of knowledge across the entire spectrum of the life sciences and the critical role played by novel technologies suggests that there are many important roles outside of academia that could be played by traditional dual-degree graduates as well as the non-traditional variants we have suggested above. Taking advantage of their strong skills as scientific generalists, they would play critical roles in overseeing product development at startups, managing portfolios of life science startups at venture capital firms, or leading data science efforts at health systems. The role of the physician-scientist training in these contexts is not necessarily to directly enable linking fundamental research in biology to patient care. Rather, it is to help manage the burgeoning institutional complexity of the life sciences by bringing significant intellectual depth that encompasses diverse facets of a biomedical education.\n\nThere are many individuals who have created non-traditional career paths building on MD or MD/PhD training without pursuing additional post-graduate experiences (Moawad, 2013). However, there may be significant value in a more systematic approach to training and encouraging such individuals. One possibility might consist of a 2–3 year program that encompasses a medical internship along with rotations with companies spanning different verticals in the life sciences. Such a post-graduate education would allow individuals to continue to strengthen their core clinical competencies while also giving them a survey of the many dimensions required to realize the bench-to-bedside vision.\n\n\nDiscussion\n\nThe creation of the integrated MD/PhD program showed tremendous vision and foresight among policymakers in the middle of the 20th century. Confronting an exploding knowledge-base and a shifting organizational landscape due to the professionalization of science, medical and scientific leaders realized that capitalizing on the full capabilities of post-WWII institutions required training generalists who could navigate the complexities of both the worlds of basic science and clinical medicine. We are in a fortunate position, therefore, to look back on over a half a century’s worth of hard data and accumulated cultural wisdom about the successes and impact of these programs. The paradigm of the discovery-invention cycle, which itself is a data-driven framework arising from decades of experience with academic and industrial science, is an ideal lens with which to re-examine the fundamental basis of physician-scientist training.\n\nAs the authors of the ARISE2 report quoted above argue, a continuum linking fundamental discovery to patient care remains a vision rather than a reality (American Academy of Arts and Sciences, 2013). Our thesis is that significantly richer analysis of the specific activities of physician-scientists is necessary to understand whether dual-degree training has contributed to linking these two domains. In addition to qualitative analysis, we argued for assembling biomedical datasets of research output and clinical data, which would allow policy researchers to investigate if there are objective “signatures” of physician-scientist training. We hope that the considerations we have identified here are, at the very least, compelling starting points for widespread discussion of this important topic, and we are eager to collaborate with others to refine and implement these ideas so as to positively impact the future of physician-scientist training.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgements\n\nWe would like to thank Venkatesh Narayanamurti, Erik Reinersten, and several anonymous referees for insightful conversations and feedback prior to submission to F1000Research.\n\n\nFootnotes\n\n1MBBS stands for Bachelor of Medicine / Bachelor of Surgery in countries where medical training is awarded an undergraduate degree.\n\n\nReferences\n\nAkabas MH, Tartakovsky I, Brass LF: The National MD--PhD Program Outcomes Study. American Association of Medical Colleges Reports. 2018.\n\nAlamri Y: The Combined Medical/PhD Degree: A Global Survey of Physician-Scientist Training Programmes. Clin Med (Lond). 2016; 16(3): 215–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmerican Academy of Arts and Sciences: ARISE II: Unleashing America’s Research & Innovation Enterprise. 2013. Reference Source\n\nAndreassen P, Christensen MK: Science in the Clinic: A Qualitative Study of the Positioning of MD-PhDs in the Everyday Clinical Setting. BMC Med Educ. 2018; 18(1): 115. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArndt BG, Beasley JW, Watkinson MD, et al.: Tethered to the EHR: Primary Care Physician Workload Assessment Using EHR Event Log Data and Time-Motion Observations. Ann Fam Med. 2017; 15(5): 419–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaregheh A, Rowley J, Sambrook S: Towards a Multidisciplinary Definition of Innovation. Management Decision. 2009; 47(8): 1323–39. Publisher Full Text\n\nBishop CM: Pattern Recognition and Machine Learning. Springer. 2006. Reference Source\n\nBoesch C: Nobel Prizes for Nuclear Magnetic Resonance: 2003 and Historical Perspectives. J Magn Reson Imaging. 2004; 20(2): 177–79. PubMed Abstract \| Publisher Full Text\n\nDaye D, Patel CB, Ahn J, et al.: Challenges and Opportunities for Reinvigorating the Physician-Scientist Pipeline. J Clin Invest. 2015; 125(3): 883–87. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeLuca GC, Ovseiko PV, Buchan AM: Personalized Medical Education: Reappraising Clinician-Scientist Training. Sci Transl Med. 2016; 8(321): 321fs2. PubMed Abstract \| Publisher Full Text\n\nDiAngi YT, Longhurst CA, Payne TH: Taming the EHR (Electronic Health Record) - There Is Hope. J Fam Med. 2016; 3(6): pii: 1072. PubMed Abstract \| Free Full Text\n\nFagerberg J, Professor of Business and Public Policy in the Walter a Haas School of Business David C Mowery, David C. Mowery, et al.:The Oxford Handbook of Innovation. Oxford University Press. 2005. Reference Source\n\nGeschwind DH, Flint J: Genetics and Genomics of Psychiatric Disease. Science. 2015; 349(6255): 1489–94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHall BH, Jaffe AB: Measuring Science, Technology, and Innovation: A Review. Report Prepared for the Panel on Developing Science, Technology, and Innovation Indicators for the Future. 2012. Reference Source\n\nHarding CV, Akabas MH, Andersen OS: History and Outcomes of 50 Years of Physician-Scientist Training in Medical Scientist Training Programs. Acad Med. 2017; 92(10): 1390–1398. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHerrera-Perez D, Haslam A, Crain T, et al.: A comprehensive review of randomized clinical trials in three medical journals reveals 396 medical reversals eLife. 2019; 8: pii: e45183. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInternational Schizophrenia Consortium, Purcell SM, Wray NR, et al.: Common Polygenic Variation Contributes to Risk of Schizophrenia and Bipolar Disorder. Nature. 2009; 460(7256): 748–52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJeffe DB, Andriole DA, Wathington HD, et al.: The Emerging Physician-Scientist Workforce: Demographic, Experiential, and Attitudinal Predictors of MD-PhD Program Enrollment. Acad Med. 2014; 89(10): 1398–1407. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohnson S: The Ghost Map: The Story of London’s Most Terrifying Epidemic and How It Changed Science, Cities, and the Modern World. Penguin. 2006. Reference Source\n\nKandiah DA: Sustainability of MBPhD Programmes. Clin Med (Lond). 2013; 13(2): 214. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKatz AA, Futter M, Mayosi BM: The intercalated BSc (Med) Honours/MB ChB and integrated MB ChB/PhD tracks at the University of Cape Town: models for a national medical student research training programme. S Afr Med J. 2014; 104(2): 111–3. PubMed Abstract\n\nMeltzer SJ: The Science of Clinical Medicine: What It Ought to Be and the Men to Uphold It. JAMA. 1909; 53(7): 508–12. Publisher Full Text\n\nMoawad H: Careers Beyond Clinical Medicine. OUP USA. 2013. Reference Source\n\nNarayanamurti V, Odumosu T, Vinsel L: RIP: The basic/applied research dichotomy. Issues Sci Technol. 2013; 29(2): 31–36. Reference Source\n\nNarayanamurti V, Odumosu T: Cycles of Invention and Discovery. 2016. Publisher Full Text\n\nNational Academies of Sciences, Engineering, and Medicine, Division of Behavioral and Social Sciences and Education, and Committee on National Statistics: Advancing Concepts and Models for Measuring Innovation: Proceedings of a Workshop. National Academies Press. 2017. Publisher Full Text\n\nNielsen M: Reinventing Discovery: The New Era of Networked Science. Princeton University Press. 2011. Reference Source\n\nO'Mara RJ, Hsu SI, Wilson DR: Should MD-PhD programs encourage graduate training in disciplines beyond conventional biomedical or clinical sciences? Acad Med. 2015; 90(2): 161–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPlavén-Sigray P, Matheson GJ, Schiffler BC, et al.: The readability of scientific texts is decreasing over time. eLife. 2017; 6: pii: e27725. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRiordan M, Hoddeson L, Herring C: The Invention of the Transistor. In More Things in Heaven and Earth. Springer. 1999; 71(2): 563–78. Publisher Full Text\n\nRoberts SF, Fischhoff MA, Sakowski SA, et al.: Perspective: Transforming science into medicine: how clinician-scientists can build bridges across research's \"valley of death\". Acad Med. 2012; 87(3): 266–70. PubMed Abstract \| Publisher Full Text\n\nRosenberg LE: MD/PhD programs--a call for an accounting JAMA. 2008; 300(10): 1208–9. PubMed Abstract \| Publisher Full Text\n\nSarma GP, Faundez V: Integrative biological simulation praxis: Considerations from physics, philosophy, and data/model curation practices. Cell Logist. 2017; 7(4): e1392400. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShanafelt TD, Dyrbye LN, Sinsky C, et al.: Relationship Between Clerical Burden and Characteristics of the Electronic Environment With Physician Burnout and Professional Satisfaction. Mayo Clin Proc. 2016; 91(7): 836–48. PubMed Abstract \| Publisher Full Text\n\nSklar DP: We Must Not Let Clinician-Scientists Become an Endangered Species. Acad Med. 2017; 92(10): 1359–1361. PubMed Abstract \| Publisher Full Text\n\nStern O: The Method of Molecular Rays. Nobel Lecture. 1946. Reference Source\n\nStossel TP: The discovery of statins. Cell. 2008; 134(6): 903–5. PubMed Abstract \| Publisher Full Text\n\nSullivan PF, Daly MJ, O'Donovan M: Genetic architectures of psychiatric disorders: the emerging picture and its implications. Nat Rev Genet. 2012; 13(8): 537–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSutton J, Killian CD: The MD-PhD researcher: what species of investigator? Acad Med. 1996; 71(5): 454–59. PubMed Abstract \| Publisher Full Text\n\nTabet JC, Rebuffat S: [Nobel Prize 2002 for chemistry: mass spectrometry and nuclear magnetic resonance]. Med Sci (Paris). 2003; 19(8–9): 865–72. PubMed Abstract \| Publisher Full Text\n\nThiele RH: Aligning Health Care and Academia: The Clinician Innovator. Acad Med. 2018; 93(7): 960–61. PubMed Abstract \| Publisher Full Text\n\nWeggemans MM, Friesen F, Kluijtmans M, et al.: Critical Gaps in Understanding the Clinician-Scientist Workforce: Results of an International Expert Meeting. Acad Med. 2019; 94(10): 1448–54. PubMed Abstract \| Publisher Full Text\n\nZemlo TR, Garrison HH, Partridge NC, et al.: The physician-scientist: career issues and challenges at the year 2000. FASEB J. 2000; 14(2): 221–30. PubMed Abstract \| Publisher Full Text" }	[ { "id": "58411", "date": "21 Jan 2020", "name": "Paul A. Randazzo", "expertise": [ "Reviewer Expertise Cancer biology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper opens two discussions. One is about the value of programs specifically designed to train physician/scientists, i.e. MD/PhD, with discussion of approaches to determine whether the programs achieve desired goals, while admitting that the goal of having a continuum of basic science and clinical research has not been achieved.\n\nThe second discussion is how to optimize training of physician/scientists, assuming, for instance, that MD/PhD programs have in place efforts to achieve the goal of the continuum of research as well as discussing other areas in which the dual degree may be valuable. Perhaps ironically, as they argue the case with the basic science and clinical training of MD/PhDs are not well integrated, neither are the two parts of this commentary.\nQuestions that would be interesting to address include how the data they propose to collect determining whether the MD/PhD programs are achieving their goals would be used to inform redesign of the programs. They are assuming shortcomings in their proposed redesign of programs, but don't have data to support that, or at least it is not well articulated. Nevertheless, the discussion is worthwhile and the integration may not be possible. The other topic that might be included is how criteria for career advancement may influence achieving the goals of the programs. Financial issues are discussed, but the criteria used to advance and retain individuals with the training are not.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "58687", "date": "11 Feb 2020", "name": "Bryan G. Yipp", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis opinion paper highlights the continued challenges faced by practicing physician-scientists, MD/PhD trainees and training programs in general. Overall, the authors highlight ongoing questions including, what is the best way to train physician-scientists? What are the roles of physician-scientists? And what are the metrics of success for physician-scientists?\n\nThe first issues raised by the authors surround training programs and specifically about the formalized MD/PhD training programs in the United States. The introduction of the discovery-invention cycle is conceptually interesting; however, their discussion presumes that the formal combined MD/PhD program is the gold standard of physician scientist training. Indeed, this is an unproven idea that formalized MD/PhD programs are the optimal means to train physician-scientists. Since the formalization of the MD/PhD and MSTP in the US there has been steady increases in the number of trainees which has not resulted in the increase in working physician researchers. Therefore, it should be considered that the current training methods may not achieve the desired goals, if the desired goals can even be agreed upon. Interestingly, the authors use Nobel prize discoveries to illustrate their point about the discovery-invention cycle, but it should be noted that many Nobel Laureates have been MD trained without a PhD degree nor completing a formal MD/PhD combined program.\nThe authors point out that a “rigid environment… would have failed to deliver on the true potential” in terms of the discovery-invention cycle; however, this same rigid environment may be having similar negative consequences to physician-scientist training. For example, there continues to be issues of diversity in the physician-scientist work force and within MD/PhD training programs. Data suggest that there are significant inequalities based on race, ethnic groups, socioeconomic status and sex. It remains uncertain if the rigid construct of MD/PhD training plays any role in these inequalities; however in our institution we have observed and published that training programs with flexible research pathways have resulted in increases in female medical doctors who work as researchers.\nAnother rigid assumption made by the authors is that “physician-scientist are meant to play the unique role of bridge builders”. The assertion that physician researchers have a predestined role in biomedical research as “translationalists” is too constraining. First, physician-scientist have had very positive impacts on discovery and mechanistic research, again exemplified by MD Nobel Laureates. Indeed, my own postdoctoral supervisor Dr. Ralph Steinman was MD trained but went on to be awarded the 2011 Nobel prize in medicine for the discovery of dendritic cells. During a conversation about translational research, Dr. Steinman asked, “Why can’t physician-scientists just be allowed to discover things?” a question that helped shape my own research career. A second comment about this issue, is that translational research has benefited tremendously from PhD scientists without formal clinical training. Therefore, translation need not be owned by physician-scientists, nor should MD researchers be boxed-in to being translational scientists.\nThe authors propose ideas to try to define what physician-scientists do and how to measure their unique impact on research and health. This includes defining metrics of success and outcomes of training programs. This is an interesting and very challenging area. Indeed, there is currently no consensus to the meaning of success. Many variables could be considered, including job position, publication numbers, grants, students, invited lectures and so on, but it is correctly pointed out that both qualitative and quantitative measures must be utilized. Despite this, there will likely never be agreement of the markers of success and this is not unique to physician-scientists but is also true for PhD scientists.\nThe opinion article ends with some interesting ideas about future training pathways which are worth considering. Indeed, the concepts of scientist-physicians or those who study medicine but do not intend to practice, is an attractive alternate pathway.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-2123
https://f1000research.com/articles/7-504/v1	26 Apr 18	{ "type": "Research Article", "title": "Non-HDL cholesterol is better than LDL-c at predicting atherosclerotic cardiovascular disease risk factors clustering, even in subjects with near-to-normal triglycerides: A report from a Venezuelan population", "authors": [ "Valmore Bermúdez", "Wheeler Torres", "Juan Salazar", "María Sofía Martínez", "Edward Rojas", "Luis Carlos Olivar", "Victor Lameda", "Ángel Ortega", "Paola Ramírez", "Milagros Rojas", "Sheena Rastogi", "Rosanna D’Addosio", "Kyle Hoedebecke", "Modesto Graterol", "Resemily Graterol", "Sandra Wilches", "Mayela Cabrera de Bravo", "Joselyn Rojas-Quintero", "Wheeler Torres", "Juan Salazar", "María Sofía Martínez", "Edward Rojas", "Luis Carlos Olivar", "Victor Lameda", "Ángel Ortega", "Paola Ramírez", "Milagros Rojas", "Sheena Rastogi", "Rosanna D’Addosio", "Modesto Graterol", "Resemily Graterol", "Sandra Wilches", "Mayela Cabrera de Bravo", "Joselyn Rojas-Quintero" ], "abstract": "Background: Non-high density lipoprotein cholesterol (non-HDL-c) has emerged as an important tool in primary prevention of atherosclerotic cardiovascular disease (ASCVD), especially among those at high risk. The main objective of this study was to evaluate the predictive value of non-HDL-c for the coexistence aggregation of multiple ASCVD risk factors and compare this with LDL-c in general subjects with normal or near normal triglycerides from Maracaibo city in Venezuela. Methods: This is a descriptive, cross-sectional study with a randomized multistage sampling. 2026 subjects were selected for this study, all were adults ≥18 years old of both genders and inhabitants of Maracaibo city, Venezuela. A complete history and physical medical assessment was performed. A multivariate logistic regression model was used to determine the odds ratio (CI95%) for the coexistence of multiple risk factors for ASCVD. Results: The median (p25-p75) of non-HDL-c was 143 mg/dL (114-174 mg/dL). 52.1% (n=1056) of the sample were women, with a median of 144 mg/dL (115-174 mg/dL) among women and 143 mg/dL (114-17 4mg/dL) among men; p=0.740. Individuals ≥50 years old, smokers, those with hypertension, obesity, diabetes, high waist circumference and elevated hs-C Reactive Protein, all had higher levels of non-HDL-c. A lower median was observed among those <30 years of age with elevated physical activity levels in their leisure time. Non-HDL-c between 130-159 mg/dL (OR=2.44; CI 95%=1.48-4.02; p<0.001) and ≥160 mg/dL (OR=3.28; CI 95%=1.72-6.23; p<0.001) was associated with greater risk of coexistent multiple risk factors for ASCVD, albeit LDL-c was not significant in the multivariate model. Conclusions: Elevated non-HDL-c was associated with conglomeration of multiple risk factors for ASCVD. This suggests evaluation of non-HDL-c may be of better utility in primary care for early identification of subjects for high risk of ASCVD. Future research might focus on the influence of non-HDL-c in cardiovascular mortality.", "keywords": [ "non-HDL-c", "LDL-c", "cholesterol", "ASCVD", "risk factor", "Coronary Artery Disease" ], "content": "Introduction\n\nAtherosclerotic cardiovascular disease (ASCVD) is the most common cause of morbidity and mortality in the world, representing 31.5% of deaths, with approximately 17.3 million deaths globally1. Hyperlipidemia plays an important role in the pathogenesis of atherosclerosis by inducing chronic inflammation, arterial plaque formation and remodeling, leading to compromised perfusion. Thankfully, hyperlipidemia remains a modifiable risk factor for ASCVD2,3.\n\nHistorically, the therapeutic goal for ASCVD risk reduction was to reduce cholesterol levels associated with low density lipoproteins (LDL-c), as elevated quantities have been associated with a higher incidence of ASCVD4. An important body of evidence, including randomized controlled trials, have demonstrated that statins reduce mortality from ASCVD when used as primary or secondary prevention5–8. Nonetheless, other studies have shown that the risk for future cardiac events remain elevated despite achieving LDL-c goals, suggesting that LDL-c might not be the best estimator of ASCVD in some populations9,10.\n\nLDL-c levels only reflect the amount of cholesterol contained within the low density lipoproteins, but does not quantify its quantity, size or structure. Additionally, there are other lipoproteins that possess atherogenic properties, such as very low density lipoproteins (VLDL-c), chylomicrons, and lipoprotein remnants. All these have Apo-B, and can participate in atherogenesis by accumulation in the intima and eliciting pro-inflammatory responses11. Other disadvantage of using LDL-c is the methodologic limitation of its calculations using Friedewald´s equation, which cannot be used in the setting of hypertriglyceridemia12. Recall that elevated triglycerides (TGs) can independently increase the risk for ASCVD13. Therefore, non-high density lipoprotein (HDL) cholesterol has emerged as an alternative predictor of ASCVD.\n\nNon-HDL cholesterol essentially represents the sum of all lipoproteins that have atherogenic properties (LDL, VLDL, IDL, lipoprotein remnant)11. Studies such as the Emerging Risk Factors Collaboration14 (N=302,430) suggest that aiming to reduce non-HDL disregarding other lipid parameters might be a new and better approach. This is supported by the fact that patients in this study with elevated non-HDL-c had higher risk of cardiac events (HR=1.50; CI 95%=1.39–1.61) than those with elevated TGs (HR=0.99; CI 95%=0.94–1.05) or with elevated LDL-c (HR=1.38; CI 95%=1.09–1.73). Moreover, non-HDL-c has demonstrated to be a useful predictor for the appearance of metabolic syndrome, which can be of great utility in primary care settings15. Lastly, non-HDL-c seems to be a better predictor of metabolic syndrome compared with LDL-c, even in patients with TG <400 mg/dL, and the predictive value was independent from central obesity and insulin resistant states16.\n\nDespite all the advantages of non-HDL-c in order to estimate ASCVD risk, the current practice measurement of non-HDL-c is underused. The objective of this study was to evaluate the predictive value of non-HDL-c for the aggregation of multiple ASCVD risk factors and compare it with LDL-c in general subjects with normal or near normal TGs from Maracaibo municipality in Venezuela.\n\n\nMethods\n\nThe Maracaibo City Metabolic Syndrome Prevalence Study (MMSPS) is a descriptive and cross-sectional study carried out by our research group in Maracaibo, Venezuela, with the main goal to determine the prevalence of metabolic syndrome in this population and it´s methodology was described previously17. For the purpose of the present sub-study, individuals with no determination of fasting insulin level were excluded; thus, a total of 2026 individuals older than 18 years old were included for this investigation. The study was approved by the Bioethics Committee of the Endocrine and Metabolic Diseases Research Center – University of Zulia (approval number: BEC-006-0305). This ethical approval included all future studies that used the data from the MMSPS. All participants signed written consent before being questioned and physically examined by a trained team.\n\nAll individuals underwent a full history and physical exam by trained personnel. During the initial interview, personal and family history of premature ASCVD, endocrine and metabolic diseases were explored. Age, gender, as well as social and economic stratus using Graffar’s scale modified by Mendez-Castellano18, were recorded. Smoking history was categorized in three different classes: a) current smoker (smoked >100 cigarettes in a lifetime, current smoking, and chronic smoker who stopped for <1 year; b) ex-smoker (smoker who stopped smoking for >1 year); c) non-smoker (never smoked or who smoked <100 cigarettes in a lifetime). Current drinkers were considered to be those having drunk >1 gram a day20.\n\nPhysical activity was assessed by the Long Form of the International Physical Activity Questionnaire (IPAQ)21. This instrument quantifies the amount of minutes invested in transportation, work, homework (gardening, cleaning), and leisure time. The participants were divided into quintiles based on total Metabolic Equivalents (METs)/min/week scores considering a sedentary person those with a MET score of 0 and those individuals with some degree of physical activity (≥1 MET) were stratified into five groups: very low (Q1), low (Q2), moderate (Q3), high (Q4) and very high (Q5) for a total of six categories. Leisure time was classified as follows: Q1 or very low PA in men<296.999 METs and women <230.999 METs; b) Q2 or low PA in men 297.000–791.999 METs and women 231.000–445.499 METs; c) Q3 or moderate PA in men 792.000–1532.399 METs and in women 445.500–742.499 METs; d) Q4 high PA in men 1532.400–2879.999 METs and in women 742.500–1798.499 METs; and e) Q5 or very high PA in men ≥2879.000 METs and women ≥1798.500 METs.\n\nBlood pressure was measured by manual methods using a sphygmomanometer and stethoscope to detect 1st and 5th Korotkoff’s sounds for systolic and diastolic blood pressure, respectively. Participants had a 15 minute resting period before BP determination, they were seating with both feet on the ground. Measurements were repeated three times in 15 minute intervals. Joint National Committee 7 (JNC7) was used to classify BP as normal BP <120/80 mmHg, prehypertension in those with systolic blood pressure (SBP) 120–139 mmHg and/or diastolic blood pressure (PAD) between 80–89 mmHg, and hypertension when BP is ≥140/90 mmHg22.\n\nHeight was determined using a calibrated stadiometer placed on a flat surface. Weight was determined using a digital scale (Tanita, TBF-310 GS Body Composition Analyzer, Tokyo – Japan), with the patient wearing light clothing and barefoot. Body mass index (BMI) was determined using Quetelec´s equation [weight/height2], and using World Health Organization criteria participants were deemed normal weight (BMI <25 kg/m2), overweight (25.0 – 29.9 kg/m2), and obese (≥30.0 kg/m2)23.\n\nWaist circumference was measured using a standardized metric belt using the metric system in centimeters and millimeters. An anatomic reference was used to measure waist circumference an equidistant point between the lower border of the ribs and the antero-superior iliac spine, according to the National Institutes of Health of the United States24. Central obesity was considered if waist circumference was ≥91 cm in women and ≥98 cm in men, according to the specific cut off values proposed for the population of Maracaibo, Venezuela25.\n\nAntecubital venous sampling was performed after an eight hour period of fasting. Samples were centrifuged and serum was obtained. Levels of glucose, total cholesterol, and TGs were determined using commercial enzymatic and colorimetric ELISA kits (Human Gesellshoft Biochemica and Diagnostica MBH). Glucose levels were interpreted according to the American Diabetes Association 2017 diagnostic criteria as follows: normal glucose <100 md/dL, impaired fasting glucose when fasting glucose is 100–125 mg/dL, and diabetes mellitus when glucose was ≥126 mg/dL26. Before diagnosing diabetes, a confirmatory test was repeated on a different day. Levels of high sensitive C reactive protein (hs-CRP) were determined using immunoturbidimetric analyses (Human Gesellshoft Biochemica and Diagnostica MBH), and the cut off point for an elevated hs-CRP was ≥0.765 mg/L27.\n\nFasting insulin concentration was determined using a commercial kit based on ELISA (DRG International. Inc. USA. New Jersey), with a detection limit of <1 mU/L. Resistance to insulin was calculate by the software HOMA-Calculator v2.2.2 provided by the Oxford Centre for Diabetes Endocrinology and Metabolism. Cutoff value for HOMA2-IR was 2.0028.\n\nNon-HDL cholesterol levels were calculated with the following formula:\n\nNon-HDL-c = total cholesterol – HDL-c\n\nLDL-c were determined using Friedwald formula29. Cutoff points for non-HDL-c: a) <130 mg/dL; b) 130–159 mg/dL; and c) ≥160 mg/dL. Cutoff points for LDL-c: a) <100 mg/dL; b) 100–129 mg/dL; and c) ≥130 mg/dL30.\n\nThe aggregation of multiple risk factors was considered when one individual presented with two or more of the following:\n\nFasting glucose ≥100 mg/dL;\n\nBlood pressure ≥130/85 mmHg;\n\nWaist circumference ≥91 cm in females and ≥98 cm in males\n\nHOMA2IR≥2.\n\nQualitative variables were shown as absolute and relative frequencies. Associations between these variables were explored using χ2 (Chi square) testing and differences with Z test. Quantitative variables were shown as arithmetic mean ± standard deviation after normality testing was performed using the Geary test. Non-normal distribution variables were logarithmically transformed and analyzed as with parametric testing when normality was achieved. When these variables remained non-normal they were shown as median with interquartile ranges (p25–p75th). U Mann Whitney test and Kruskal-Wallis test were used for comparisons between two groups and three or more groups, respectively.\n\nA multivariate regression model was created to estimate odds ratio and confidence intervals for prediction of composite of multiple risk factors. The first model was adjusted for age, sex, age group, ethnic group, socio-economic status, literacy, employment status, smoking, alcohol consumption, physical activity during leisure time, hypertension, hs-CRP, LDL-c and non-HDL cholesterol.\n\nSPSS v.21 for Windows (IBM Chicago, IL) was used for statistical analyses and data gathering. We considered results statistically significant at p<0.05.\n\n\nResults\n\nFrom the 2026 participants, 52.1% (n=1056) were female and 47.9% were male (n=846). The mean age was 40.79±15.76 years. Other general features are presented in Table 1. Median non-HDL-c was 143 mg/dL (114–174) mg/dL, with 144 (115–174) mg/dL among females and 143 (114–174) mg/dL in males; p=0.740.\n\nAll results are shown as arithmetic mean and standard deviation. except Non HDL-c (median p25–p75th).\n\nAbbreviations: HDL-c: High density lipoprotein cholesterol; LDL-c: Low density lipoprotein cholesterol; HOMA: Homeostasis model assessment.\n\nTable 2 shows the epidemiology of non-HDL-c according to social and demographic features. Non-HDL-c levels showed an increasing trend with age, from 118 (97–143) mg/dL in those <30 years old, 151 (124–175) mg/dL among those from 30–49 years old and 166 (137–196) mg/dL in >50 years old; p<0.001. On the other hand, indigenous Venezuelan populations showed lower non-HDL-c levels (127; 97–151 mg/dL) compared with mixed race (145; 116–175 mg/dL) and white Hispanics (145; 114–176 mg/dL; p<0.001).\n\n* Mann-Whitney U Test; for 3 or more categories: Kruskal-Wallis H test.\n\n§ Positive alcohol consumption: ≥1 gram/day\n\nHigher levels of non-HDL-c were found among smokers (151; 118–183 mg/dL) compared with non-smokers or ex-smokers, p=0.001. Subjects with very high physical activity exhibited lower non-HDL-c levels 124 (98–160) mg/dL when compared with sedentary subjects [147 (118–175) mg/dL; p<0.001]. No significant differences were found when comparing alcohol drinkers and non-drinkers.\n\nTable 3 shows non-HDL-c levels according to clinical, metabolic, and anthropometric variables. Non-HDL-c were significantly higher among those with hypertension compared to those with normal blood pressure (159 vs. 132 mg/dL, respectively; p<0.001). This behavior was also observed when comparing obese and normal weight individuals (155 vs. 124 mg/dL, respectively; p<0.001), type 2 diabetes and non-diabetic individuals (161 vs. 137 mg/dL; p<0.001), abdominal obesity and persons with normal waist circumference (154 vs. 132 mg/dL; p<0.001), and elevated hs-CRP vs. normal hs-CRP (156 vs. 140 mg/dL; p<0.001). Tertile distribution according non-HDL-c and both, clinical and anthropometric variables are shown in Table 4.\n\nAbbreviations: BMI: Body mass index; BP: Blood pressure; hsCRP: High-sensitivity C reactive proteína; JNC-7: The Seventh Report of the Joint National Committee on hypertension.\n\n† Cutoff for Maracaibo adult population: ≥98 cm for men and ≥91 cm for women).\n\n§ American Diabetes Association (ADA) blood glucose diagnostic criteria.\n\n*Mann-Whitney U test; for 3 or more categories: Kruskal-Wallis H test.\n\nAbbreviations: BMI: Body mass index; BP: Blood pressure; hsCRP: High-sensitivity C reactive proteína; JNC-7: The Seventh Report of the Joint National Committee on hypertension.\n\n† Cutoff for Maracaibo adult population: ≥98 cm for men and ≥91 cm for women).\n\n§ American Diabetes Association (ADA) blood glucose diagnostic criteria.\n\nFigure 1 shows levels of non-HDL-c according to the number of risk factors for ASCVD. Those with any risk factor had a non-HDL-c of 122 (98–146) mg/dL, and 161 (131–192) mg/dL in those with three criteria and 159 (137–195) mg/dL in those with four criteria; p<0.001. Table 5 shows a multivariate logistic regression model where levels of non-HDL-c between 130 – 159 mg/dL, (OR=2.59; CI95%: 1.62-4.13; p<0.001) and ≥160 mg/dL (OR=3.75; CI95%=2.04-6.91; p<0.001), had an inverse probability of presenting a composite of multiple risk factors, while LDL-C was not significantly associated (OR=0.42; CI95%: 0.23-0,95; p=0.035).\n\nKruskal-Wallis H Test: p<0.001.\n\na CI: Confidence Interval at 95%.\n\nb Significance level\n\nc Model 1 Adjusted by: sex, age groups, ethnicity, social-economic status, educative status, marital status, working status, smoking habits, alcohol consumption, physical activity in Leisure time dominion, hsCRP, LDL-c and No-HDL-c.\n\n\nDiscussion\n\nFor nearly 50 years, incredible efforts have been made to identify specific and prevalent ASCVD risk factors, planning and application of primary and secondary prevention strategies, evaluation of population genetics and overall ethnicity genetic risks, and modification due to epigenetics. These risk factors have been of various natures, from anthropometric measurements, such as BMI and waist circumference, lifestyle patterns, to blood lipids sub-fractions, such as LDL-c and HDL-c. In regards to the focus of the present study, lipid profiles and novel lipid fractions and their association with ASCVD have been the main focus of grand scale epidemiological, clinical, and pharmacological investigation31,32.\n\nIn spite of all the efforts, data has been accumulating that suggests that focusing on one lipid fraction, namely LDL-c, may not be the appropriate approach33, due to recently described atherogenic particles, like IDL, Apo B, and non-HDL33. The concept of cardiovascular residual risk factor has been intimately associated with cardiovascular disease reduction, being twice as effective as LDL-c34. In fact, Helgadottir et al.35 reported that genetic risk scores using non-HDL-c strongly associates with coronary artery disease, and this genetic risk was considerably lower than that offered by LDL-c. It is no coincidence that non-HDL-c has been shown to correlate with coronary artery disease progression, cardiovascular morbidity, and mortality34,36.\n\nThe present results show that higher non-HDL-c levels were associated with higher risk of multiple risk factors for ASCVD. These results are similar to those reported by Kumar et al. where non-HDL-c had a better predictive value than LDL-c for atherosclerosis among those with TGs >150 mg/dl37. This study excluded patients with increased TGs >400 mg/dl; therefore, one cannot assume this association is also seen in this group. Moreover, Arsenault et al.38 followed over 21 thousand subjects without diabetes or previous coronary heart disease (CHD), demonstrating that high non-HDL-c is associated with increased CHD.\n\nFollowing the recommendation of the Strong Heart Study39, the recent 2016 ACC Expert Consensus Decision Pathway on the Role of Non-Statin Therapies for LDL-Cholesterol Lowering in the Management of Atherosclerotic Cardiovascular Disease Risk proposed a goal of <100 mg/dl for non-HDL-c in diabetic patients40. As expected, subjects with diabetes in our population have higher non-HDL-c, which is a recognized risk factor in diabetic subjects at risk for ASCVD41. Interestingly, Apo B and non-HDL-c are better predictors of diabetes development than glycated hemoglobin42. In line with this notion, the present results also show that non-HDL-c is associated with higher levels of hs-CRP (systemic inflammation), hypertension, and central obesity. We previously described our population as having a high prevalence of obesity and overweight, managing a staggering 65.7%43. Thus, the overlapping of risk factors and metabolic syndrome/type 2 diabetes development is imminent and borderline epidemic.\n\nLastly, Hispanic population seems to be at higher risk for LDL-particle numbers and non-HDL-c discordance44. Kilgore et al.45 reported that subjects with high non-HDL-c and normal LDL-c were likely to be Hispanic males with metabolic syndrome and other cardiovascular risks. Likewise, using the database from The Hispanic Community Health Study/Study of Latinos, Rodriguez et al.46 reported that almost two thirds of Latinos have a form of dyslipidemia, with South Americans having high non-HDL-c and high LDL-c. Therefore, ethnicity is of high importance when evaluating clinical risk for ASCVD, including blood lipid profiles and sedentary lifestyles in these groups47.\n\nTo summarize, this investigation in Hispanic population shows that non-HDL-c is associated with multiple risk aggregation for ASCVD, being associated with hypertension, central obesity and low grade inflammation. The question that arises is: Should non–HDL-c replace LDL-C as the main target of therapy?33. The fact that non–HDL-c is a better risk predictor, can be performed in a non-fasting state, and can be easily calculated by extracting HDL-c from total cholesterol without using any other laboratory assay makes it the most advantageous parameter for prediction of ASCVD even in subjects with TAG <200 mg/dl.\n\n\nData availability\n\nDataset 1: MMSPS non-HDL and atherosclerotic cardiovascular disease risk factors raw data. DOI, 10.5256/f1000research.13005.d19598048", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Technological, Humanistic, and Scientific Development Council (Consejo de Desarrollo Científico, Humanístico y Tecnológico; CONDES), University of Zulia (grant nº CC-0437-10-21-09-10).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nRoth GA, Huffman MD, Moran AE, et al.: Global and Regional Patterns in Cardiovascular Mortality From 1990 to 2013. Circulation. 2015; 132(17): 1667–1678. PubMed Abstract \| Publisher Full Text\n\nPasterkamp C, Falk E: Atherosclerotic plaque rupture: an overview. Journal of Clinical and Basic Cardiology. 2000; 3(2): 81–6. Reference Source\n\nBoyle JJ: Macrophage activation in atherosclerosis: pathogenesis and pharmacology of plaque rupture. Curr Vasc Pharmacol. 2005; 3(1): 63–8. PubMed Abstract \| Publisher Full Text\n\nKannel WB, Dawber TR, Thomas HE Jr, et al.: Comparison Of Serum Lipids In The Prediction Of Coronary Heart Disease. Framingham Study Indicates That Cholesterol Level And Blood Pressure Are Major Factors In Coronary Heart Disease; Effect Of Obesity And Cigarette Smoking Also Noted. R I Med J. 1965; 48: 243–50. PubMed Abstract\n\nScandinavian Simvastatin Survival Study Group: Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet. 1994; 344(8934): 1383–9. PubMed Abstract \| Publisher Full Text\n\nLong-Term Intervention with Pravastatin in Ischaemic Disease (LIPID) Study Group: Prevention of cardiovascular events and death with pravastatin in patients with coronary heart disease and a broad range of initial cholesterol levels. N Engl J Med. 1998; 339(19): 1349–57. PubMed Abstract \| Publisher Full Text\n\nThe Heart Protection Study Collaborative Group: MRC/BHF heart protection study of cholesterol lowering with simvastatin in 20,536 high-risk individuals: a randomized placebo-controlled trial. ACC Curr J Rev. 2002; 11(6): 34–5. Publisher Full Text\n\nWillerson JT: Effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. Circulation. 1996; 94(12): 3054. PubMed Abstract \| Publisher Full Text\n\nJepsen AM, Langsted A, Varbo A, et al.: Increased Remnant Cholesterol Explains Part of Residual Risk of All-Cause Mortality in 5414 Patients with Ischemic Heart Disease. Clin Chem. 2016; 62(4): 593–604. PubMed Abstract \| Publisher Full Text\n\nSampson UK, Fazio S, Linton MF: Residual cardiovascular risk despite optimal LDL cholesterol reduction with statins: the evidence, etiology, and therapeutic challenges. Curr Atheroscler Rep. 2012; 14(1): 1–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFruchart JC, Davignon J, Hermans MP, et al.: Residual macrovascular risk in 2013: what have we learned? Cardiovasc Diabetol. 2014; 13(1): 26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFukuyama N, Homma K, Wakana N, et al.: Validation of the Friedewald Equation for Evaluation of Plasma LDL-Cholesterol. J Clin Biochem Nutr. 2008; 43(1): 1–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBermúdez V, Salazar J, Calvo M, et al.: Importance of high triglycerides levels between novel coronary risk factors. Revista Colombiana de Cardiología. 2017; 24(6): 583–591. Publisher Full Text\n\nThe Emerging Risk Factors Collaboration, Di Angelantonio E, Sarwar N, et al.: Major lipids, apolipoproteins, and risk of vascular disease. JAMA. 2009; 302(18): 1993–2000. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGhodsi S, Meysamie A, Abbasi M, et al.: Non-high-density lipoprotein fractions are strongly associated with the presence of metabolic syndrome independent of obesity and diabetes: a population-based study among Iranian adults. J Diabetes Metab Disord. 2017; 16: 25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoriyama K, Takahashi E: Non-HDL Cholesterol is a More Superior Predictor of Small-Dense LDL Cholesterol than LDL Cholesterol in Japanese Subjects with TG Levels <400 mg/dL. J Atheroscler Thromb. 2016; 23(9): 1126–37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBermúdez V, Marcano RP, Cano C, et al.: The Maracaibo city metabolic syndrome prevalence study: design and scope. Am J Ther. 2010; 17(3): 288–94. PubMed Abstract \| Publisher Full Text\n\nMéndez-Castellano H, De Méndez M: Estratificación social y biología humana: método de Graffar modificado. Arch Venez Pueric Pediatr. 1986; 49: 93–104. Reference Source\n\nBermúdez V, Miquilena E, Salazar J, et al.: Smoking Habit in Adult Population from Maracaibo City, Venezuela. Int J Respir Pulm Med. 2016; 3(6): 61. Publisher Full Text\n\nBermúdez V, Torres Y, Apruzzese V, et al.: Alcohol drinking patterns in the adult population from the Maracaibo municipality, Zulia – Venezuela. Revista Latinoamericana de Hipertensión. 2014; 9(3): 21–8. Reference Source\n\nInternational Physical Activity Questionnaire (IPAQ) and New Zealand Physical Activity Questionnaire (NZPAQ): A doubly labelled water validation. J Sci Med Sport. 2007; 10(1): 52. Publisher Full Text\n\nChobanian AV, Bakris GL, Black HR, et al.: The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure: the JNC 7 report. JAMA. 2003; 289(19): 2560–71. PubMed Abstract \| Publisher Full Text\n\nWHO: Obesity: preventing and managing the global epidemic. Report of a WHO Consultation. World Health Organ Tech Rep Ser. 2000; 894: i–xii, 1–253. PubMed Abstract\n\nHealth Statistics: NHANES III reference manuals and reports (CDROM). Hyattsville, MD: Centers for Disease Control and Prevention. 1996. Reference Source\n\nBermúdez V, Rojas J, Salazar J, et al.: Sensitivity and Specificity Improvement in Abdominal Obesity Diagnosis Using Cluster Analysis during Waist Circumference Cut-Off Point Selection. J Diabetes Res. 2015; 2015: 1–14, 750265. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmerican Diabetes Association: 2. Classification and Diagnosis of Diabetes. Diabetes Care. 2017; 40(Suppl 1): S11–S24. PubMed Abstract \| Publisher Full Text\n\nBermúdez V, Cabrera M, Mendoza L, et al.: High-sensitivity c-Reactive protein epidemiological behavior in adult individuals from Maracaibo, Venezuela. Revista Latinoamericana de Hipertensión. 2013; 8(1): 22–29. Reference Source\n\nBermúdez V, Rojas J, Martínez MS, et al.: Epidemiologic Behavior and Estimation of an Optimal Cut-Off Point for Homeostasis Model Assessment-2 Insulin Resistance: A Report from a Venezuelan Population. Int Sch Res Notices. 2014; 2014: 1–10, 616271. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFriedewald WT, Levy RI, Fredrickson DS: Estimation of the Concentration of Low-Density Lipoprotein Cholesterol in Plasma, Without Use of the Preparative Ultracentrifuge. Clin Chem. 1972; 18(6): 499–502. PubMed Abstract\n\nJellinger PS, Handelsman Y, Rosenblit PD, et al.: AMERICAN ASSOCIATION OF CLINICAL ENDOCRINOLOGISTS AND AMERICAN COLLEGE OF ENDOCRINOLOGY GUIDELINES FOR MANAGEMENT OF DYSLIPIDEMIA AND PREVENTION OF CARDIOVASCULAR DISEASE - EXECUTIVE SUMMARYComplete Appendix to Guidelines available at http://journals.aace.com. Endocr Pract. 2017; 23(4): 479–497. PubMed Abstract \| Publisher Full Text\n\nGrundy SM, Cleeman JI, Merz CN, et al.: Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III guidelines. Circulation. 2004; 110(2): 227–39. PubMed Abstract \| Publisher Full Text\n\nStone NJ, Robinson JG, Lichtenstein AH, et al.: 2013 ACC/AHA guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2014; 63: 2889–934. PubMed Abstract \| Publisher Full Text\n\nHarper CR, Jacobson TA: Using apolipoprotein B to manage dyslipidemic patients: time for a change? Mayo Clin Proc. 2010; 85(5): 440–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobinson JG, Wang S, Smith BJ, et al.: Meta-analysis of the relationship between non-high-density lipoprotein cholesterol reduction and coronary heart disease risk. J Am Coll Cardiol. 2009; 53(4): 316–22. PubMed Abstract \| Publisher Full Text\n\nHelgadottir A, Gretarsdottir S, Thorleifsson G, et al.: Variants with large effects on blood lipids and the role of cholesterol and triglycerides in coronary disease. Nat Genet. 2016; 48(6): 634–9. PubMed Abstract \| Publisher Full Text\n\nBittner V: Non-HDL Cholesterol: Measurement, Interpretation and Significance. John Hopkins Advanced Studies in Medicine. 2007; 7(1): 8–11. Reference Source\n\nKumar BV, Guntakalla YR, Thomas Z, et al.: Role of Non High Density Lipoprotein Cholesterol (Non HDL-C) in Predicting Coronary Artery Disease. Indian Journal of Pharmacy Practice. 2015; 8(4): 166–170. Reference Source\n\nArsenault BJ, Rana JS, Stroes ES, et al.: Beyond low-density lipoprotein cholesterol: respective contributions of non-high-density lipoprotein cholesterol levels, triglycerides, and the total cholesterol/high-density lipoprotein cholesterol ratio to coronary heart disease risk in apparently healthy men and women. J Am Coll Cardiol. 2009; 55(1): 35–41. PubMed Abstract \| Publisher Full Text\n\nLu W, Resnick HE, Jablonski KA, et al.: Non-HDL cholesterol as a predictor of cardiovascular disease in type 2 diabetes: the strong heart study. Diabetes Care. 2003; 26(1): 16–23. PubMed Abstract \| Publisher Full Text\n\nWriting Committee, Lloyd-Jones DM, Morris PB, et al.: 2016 ACC Expert Consensus Decision Pathway on the Role of Non-Statin Therapies for LDL-Cholesterol Lowering in the Management of Atherosclerotic Cardiovascular Disease Risk: A Report of the American College of Cardiology Task Force on Clinical Expert Consensus Documents. J Am Coll Cardiol. 2016; 68(1): 92–125. PubMed Abstract \| Publisher Full Text\n\nLin FJ, Tseng WK, Yin WH, et al.: Residual Risk Factors to Predict Major Adverse Cardiovascular Events in Atherosclerotic Cardiovascular Disease Patients with and without Diabetes Mellitus. Sci Rep. 2017; 7(1): 9179. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHwang YC, Ahn HY, Park SW, et al.: Apolipoprotein B and non-HDL cholesterol are more powerful predictors for incident type 2 diabetes than fasting glucose or glycated hemoglobin in subjects with normal glucose tolerance: a 3.3-year retrospective longitudinal study. Acta Diabetol. 2014; 51(6): 941–6. PubMed Abstract \| Publisher Full Text\n\nBermúdez V, Pacheco M, Rojas J, et al.: Epidemiologic behavior of obesity in the Maracaibo City metabolic syndrome prevalence study. PLoS One. 2012; 7(4): e35392. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDegoma EM, Davis MD, Dunbar RL, et al.: Discordance between non-HDL-cholesterol and LDL-particle measurements: results from the Multi-Ethnic Study of Atherosclerosis. Atherosclerosis. 2013; 229(2): 517–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKilgore M, Muntner P, Woolley JM, et al.: Discordance between high non-HDL cholesterol and high LDL-cholesterol among US adults. J Clin Lipidol. 2014; 8(1): 86–93. PubMed Abstract \| Publisher Full Text\n\nRodriguez CJ, Daviglus ML, Swett K, et al.: Dyslipidemia patterns among Hispanics/Latinos of diverse background in the United States. Am J Med. 2014; 127(12): 1186–94.e1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang Z, Manichukal A, Goff DC Jr, et al.: Genetic associations with lipoprotein subfraction measures differ by ethnicity in the multi-ethnic study of atherosclerosis (MESA). Hum Genet. 2017; 136(6): 715–726. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBermúdez V, Torres W, Salazar J, et al.: Dataset 1 in: Non-HDL cholesterol is better than LDL-c at predicting atherosclerotic cardiovascular disease risk factors clustering, even in subjects with near-to-normal triglycerides: A report from a Venezuelan population. F1000Research. 2018. Data Source" }	[ { "id": "33526", "date": "30 May 2018", "name": "Chau-Chung Wu", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study showed elevated non-HDL-c was associated with conglomeration of multiple risk factors for ASCVD. The result is predictable and not novel. It has been shown in many previous publications. However, one major concern about the methodology: Were the blood pressure and sugar measured before any treatment or just on treatment? The authors should clarify it, because it may change the risk calculation in some patients, esp. for those already with hypertension or diabetes mellitus from the beginning of the study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "49038", "date": "10 Jun 2019", "name": "Chandrakala Aluganti Narasimhulu", "expertise": [ "Reviewer Expertise Cardiovascular disease" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present study “Non-HDL cholesterol is better than LDL-c at predicting atherosclerotic cardiovascular disease risk factors clustering, even in subjects with near-to-normal triglycerides: A report from a Venezuelan population” by Bermúdez et al., is interesting however, the authors must address the following key points:\nIn results, (Table 2) the non-HDL cholesterol levels were represented based on age groups. In addition to this if the authors provide the analysis for men and women separately which will be more accurate. Table 3 to 5: if the analysis done for both men and women separately that provides more insights for both clinicians and researchers. Authors are suggested to comment/discuss briefly the hormonal influence in disease progression in both genders.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-504
https://f1000research.com/articles/8-2119/v1	18 Dec 19	{ "type": "Brief Report", "title": "Null effect of circulating sphingomyelins on risk for breast cancer: a Mendelian randomization report using Breast Cancer Association Consortium (BCAC) data.", "authors": [ "Charleen D. Adams" ], "abstract": "Background: Changes in cellular metabolism are a hallmark of cancer and are linked with sphingolipid synthesis. Due to immense interest in how sphingolipids influence chemoresistance, more is known about the impact of sphingolipids during cancer treatment and progression than about the potential role of sphingolipids in the induction of tumors in humans. Methods: Because estrogen triggers sphingolipid signaling cascades, the causal role of circulating levels of sphingomyelin (a type of sphingolipid) on breast cancer was investigated with a well-powered Mendelian randomization design. Results: The results reveal a null effect (OR = 0.94; 95% CI = 0.85, 1.05; P = 0.30). Conclusion: Despite the role sphingomyelins play during chemoresistance and cancer progression, circulating sphingomyelins do not appear to initiate or protect from breast cancer. This finding comprises the first causal report in humans that sphingomyelins on breast cancer initiation is null. Future investigations of risk in other cancer types are needed to further explore the potential role of sphingolipid biology in cancer etiology.", "keywords": [ "Mendelian randomization", "breast cancer", "sphingomyelins", "lipids", "metabolism" ], "content": "Introduction\n\nChanges in cellular metabolism are a hallmark of cancer1. Sphingolipids can control the rate of cell proliferation during malignant transformation and affect chemoresistance2. Sphingomyelin is a type of sphingolipid, a class of lipids containing sphingoid bases (Figure 1). As a response to cellular stress, sphingolipids mediate apoptosis and autophagy, through the synthesis and/or accumulation of ceramide. Ceramide can be hydrolyzed from sphingomyelin3. Due to immense interest in how sphingolipids influence chemoresistance, much is known about the impact of sphingolipids on cancer treatment and little is known about role sphingolipids in the induction of tumors in humans.\n\nThe bolder print indicates the lipidic sphingoid base that is carrying a saturated fatty acid amine bonded to an amino group at the C2 position. Attached to the polar head is a phosphocholine. The cartoon has been reproduced with permission from Holthuis et al. (2001)6.\n\nEstrogen triggers sphingolipid signaling cascades2. Due to this, it was hypothesized that circulating sphingomyelins might be involved in acquisition of breast cancer. The causal impact of circulating levels of sphingomyelins on risk for breast cancer was appraised with Mendelian randomization (MR).\n\n\nMethods\n\nMR is an instrumental variables technique; i.e., genetic variants (typically single-nucleotide polymorphisms, SNPs) strongly associated with traits are used in statistical models instead of the traits themselves. This avoids most environmental sources of confounding and averts reverse causation, which preclude causal inference in observational studies. Two-sample MR is an adaptation of the procedure that uses summary statistics from two genome-wide association (GWA) studies4.\n\nMR depends on the validity of three assumptions: (i) the SNPs acting as the instrumental variables must be strongly associated with the exposure; (ii) the instrumental variables must be independent of confounders of the exposure and the outcome; and (iii) the instrumental variables must be associated with the outcome only through the exposure.\n\nStep 1. Kettunen et al. (2016) performed a genome-wide association (GWA) study of 123 circulating metabolites—including sphingomyelins—in European participants (n=13,476 for sphingomyelins)5. From this, independent (those not in linkage disequilibrium; R2 < 0.01) SNPs associated at genome-wide significance (P < 5 × 10-8) with a standard-deviation (SD) increase in circulating sphingomyelins were identified. The Kettunen GWA is available through MR-Base5.\n\nStep 2. A publicly available GWA study of breast cancer performed by the Breast Cancer Association Consortium (BCAC) on 122,977 breast cancer cases and 105,974 controls of European ancestry was chosen as the outcome GWA for breast cancer7.\n\nA seven-SNP instrument for circulating sphingomyelins was constructed from the SNPs strongly associated with circulating sphingomyelin levels. Estimates of the proportion of variance in circulating sphingomyelins explained by the genetic instrument (R2) and the strength of the association between the genetic instrument and sphingomyelins (F-statistic) were generated (conventionally F-statistics <10 are weak). The instrument for sphingomyelins has an R2 = 0.032 and the F-statistic = 1089. The study was powered using the mRnd MR power calculator (available at http://cnsgenomics.com/shiny/mRnd/). It had >90% power to detect an OR of 0.90.\n\nThe log-odds for breast cancer per SD increase in circulating sphingomyelins was calculated, using the inverse-variance weighted (IVW) MR method. The “TwoSampleMR” package4 was used for the MR analysis.\n\nAll described analyses were performed in R version 3.5.2.\n\nSeveral sensitivity estimators can be used appraise pleiotropic bias. Three were chosen to complement the primary IVW causal tests: MR Egger regression, weighted median, and weighted mode estimations. In addition to these sensitivity estimators, a test for heterogeneity was performed, since variability in the causal estimates between SNPs can indicate pleiotropy. The test for heterogeneity was performed using Cochrane’s Q-statistic.\n\n\nResults\n\nThere was a null effect for circulating sphingomyelins on breast cancer (OR = 0.94; 95% CI = 0.85, 1.05; P = 0.30). The sensitivity estimators had effect estimates in the same direction and were of comparable magnitude to the IVW estimate, indicating no evidence for substantial bias due to unwanted pleiotropy. There was no evidence for heterogeneity in the estimates (Table 1). The MR-Egger intercept test, which provides an assessment of potential directional pleiotropy in the IVW was null. A null effect indicates a lack of evidence for pleiotropy (Estimate = 1.01; 95% CI = 0.97, 1.04; P =0.55).\n\nIVW, inverse-weighted variance. *Denotes a sensitivity estimator.\n\n\nDiscussion\n\nThis is the first causal report in humans that sphingomyelins on breast cancer initiation is null. The null effect might reflect the complex interplay of pro-apoptotic and pro-growth ceramides8, perhaps with greater upregulation of the pro-apoptotic pathways, which may be different for different tissues. Future investigations of risk in other cancer types are needed to further explore the potential role of sphingolipid biology in cancer etiology.\n\nOne potential limitation of this analysis is that unwanted pleiotropy cannot be entirely ruled out in MR studies. However, the sensitivity estimators provide evidence against this. Given the many ways in which a finding could be a false-positive, null findings from well-powered MR studies are, in some ways, more believable than reports of causal associations9. A major strength of this two-sample MR analysis is that it capitalized on the power of very large GWA studies. If sphingomyelins were causal for breast cancer initiation, it is highly unlikely that the effect would go undetected with more than 100,000 cases and 100,000 controls in BCAC.\n\n\nData availability\n\nThe sphingomyelin data are publicly available through the MR-Base repository (http://www.mrbase.org/) under a GNU General Public License v3.\n\nThe breast cancer outcome data are freely available on the BCAC website (http://bcac.ccge.medschl.cam.ac.uk/).", "appendix": "References\n\nHanahan D, Weinberg RA: Hallmarks of cancer: the next generation. Cell. 2011; 144(5): 646–674. PubMed Abstract \| Publisher Full Text\n\nSukocheva O, Wadham C: Role of sphingolipids in oestrogen signalling in breast cancer cells: an update. J Endocrinol. 2014; 220(3): R25–35. PubMed Abstract \| Publisher Full Text\n\nOgretmen B: Sphingolipid metabolism in cancer signalling and therapy. Nat Rev Cancer. 2018; 18(1): 33–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHemani G, Zheng J, Elsworth B, et al.: The MR-Base platform supports systematic causal inference across the human phenome. eLife. 2018; 7: pii: e34408. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKettunen J, Demirkan A, Würtz P, et al.: Genome-wide study for circulating metabolites identifies 62 loci and reveals novel systemic effects of LPA. Nat Commun. 2016; 7: 11122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHolthuis JC, Pomorski T, Raggers RJ, et al.: The organizing potential of sphingolipids in intracellular membrane transport. Physiol Rev. 2001; 81(4): 1689–1723. PubMed Abstract \| Publisher Full Text\n\nMichailidou K, Lindström S, Dennis J: Association analysis identifies 65 new breast cancer risk loci. Nature. 2017; 551(7678): 92–94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPralhada Rao R, Vaidyanathan N, Rengasamy M: Sphingolipid metabolic pathway: an overview of major roles played in human diseases. J Lipids. 2013; 2013: 178910. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVanderWeele TJ, Tchetgen Tchetgen EJ, Cornelis M, et al.: Methodological challenges in mendelian randomization. Epidemiology. 2014; 25(3): 427–435. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "61188", "date": "23 Mar 2020", "name": "Jorge Simon", "expertise": [ "Reviewer Expertise Hepatology", "metabolomics", "lipidomics", "sphingolipids" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI consider this manuscript suitable for indexing with your journal with some changes recommended:\nMake a more user-friendly introduction and be sure about some concepts (e.g sphingomyelin is a type of sphingolipid containing sphingomyelin, where every sphingolipid contains it).\n\nIntroduce some main concepts about breast concept to contextualize your research.\n\nIn the part of results, include what does each variable mean. I have found difficult to understand all the data from the table and the reader could have the same \"problem\".\n\nIn the part of discussion you should focus on the possible explanation about the crosstalk between proliferative and pro-apoptotic SPHINGOLIPIDS (not ceramides, please confirm every concept (e.g. sphingomyelin is proliferative while ceramide apoptotic, and C1P pro-apoptotic). I would like to read a little bit more about your hypothesis instead of the limitations of your study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "66881", "date": "27 Jul 2020", "name": "Rezvan Esmaeili", "expertise": [ "Reviewer Expertise cancer", "biomarkers" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscripts, authors used Mendelian randomization to determine the role of circulating sphingomyelin in breast cancer. Although the manuscript is worth indexing, the following issues should be addressed.\nThe introduction needs more explanation about breast cancer, the possible role of sphingomyelin, and MR for the general readers of the journal.\n\nHow the second and third MR assumptions (which they mentioned in the manuscript) were met?\n\nAs the authors said, the sphingomyelin is affected by estrogen signaling. Why did authors not do the subgroup analysis and not investigated the MR assumption in the ER-positive vs. negative patients? It seems the result may be significant in the ER-positive group. This analysis is necessary.\n\nA similar publication in the same data set (10.2217/bmt-2020-00021) is available. Please explain the novelty or differences of the recent manuscript with the mentioned paper? Why is it not in the references?\n\nThe authors should provide a table containing a summary of the statistical data. Mentioned the selected SNP, allele frequency, hazard radio, p values, etc.\n\nSurvival analysis should be included.\n\nA table of demographic data and clinicopathologic characteristics of patients should be included.\n\nIt seems more data should be included in regards to sensitivity analysis and power estimate. I am not an expert in this field, and another person should revise this part.\n\nThe authors should mention the previous studies related to sphingomyelin and breast cancer in the discussion.\n\nThe body of the manuscript needs some English polishing. The text should be more fluent to read.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-2119
https://f1000research.com/articles/8-1008/v1	04 Jul 19	{ "type": "Research Article", "title": "The protective effects of antigen-specific IgY on pyocyanin-treated human lymphoma Raji cells", "authors": [ "Heni Susilowati", "Sidna Artanto", "Heribertus Dedy Kusuma Yulianto", "Wihaskoro Sosroseno", "Suryani Hutomo", "Sidna Artanto", "Heribertus Dedy Kusuma Yulianto", "Wihaskoro Sosroseno", "Suryani Hutomo" ], "abstract": "Background: Pyocyanin (PCN), a highly pathogenic pigment produced by Pseudomonas aeruginosa, induces caspase 3-dependent human B cell (Raji cells) death. The aim of the present study, therefore, was to assess whether antigen-specific IgY antibodies may be protective on PCN-induced Raji cell death. Methods: Chickens were subcutaneously immunized with Freund's complete adjuvant containing PCN, and then given two boosted immunizations. Anti-PCN IgY antibodies were purified from egg yolk and detected using an agar gel precipitation test (AGPT) and ELISA. Protective effects of antigen-specific IgY on Raji cells were tested using a cell viability assay. Results: AGPT results showed the formation of strong immune complex precipitates, whilst ELISA further confirmed the presence of IgY antibodies specific to PCN at significant concentration. Further experiments showed that anti-PCN IgY antibodies significantly increased PCN-treated Raji cell viability in a dose-dependent fashion (p<0.05). Conclusions: The results of the present study suggest that anti-PCN IgY antibodies may be protective on PCN-induced Raji cell death.", "keywords": [ "Pseudomonas aeruginosa", "pyocyanin", "IgY", "protective effect" ], "content": "Introduction\n\nPseudomonas aeruginosa, an opportunistic Gram-negative bacterium, is found in the environment with a broad spectrum of habitats and is responsible for severe nosocomial infections in the urinary tract1, the respiratory tract2, the vascular system3 and the central nervous system4. It is known for one of the most common pathogens infecting patients with cystic fibrosis, leading to increase its morbidity and mortality due to the resisting abilities of this pathogen to against antibiotic treatments5,6. The presence of P. aeruginosa in dental pulp and periapical lesions may cause failure of endodontic treatments7,8. In the initial stage of infection, P. aeruginosa releases various virulent mediators, such as elastases, proteases, exotoxin A, and pyocyanin (PCN), after which chronic infection and persistent bacterial colonization at the P. aeruginosa-infected sites would be established9. PCN, a blue redox-active secondary metabolite and a member of tricyclic phenazine family, is known to function as a gene controller during the stationary growth phase10, an antibiotic11, an electron transfer facilitator12, and a potent mammary cell-damaging virulence factor13. Reports indicate that PCN inhibits B cell, T cell and macrophage functions14,15 and induces neutrophil apoptosis16, suggesting that PCN suppresses both innate and antigen-specific adaptive immune response.\n\nThe existence of multidrug-resistant (MDR) P. aeruginosa leads to the development of alternative treatment strategies to eradicate an established chronic P. aeruginosa infection. Of these treatments, both active and passive immunotherapies have been reported. Active immunization with P. aeruginosa-derived flagella in cystic fibrosis patients resulted in increased serum antigen-specific IgG antibodies and reduced number of P. aeruginosa strains, suggesting the reduction of P. aeruginosa infection risk in cystic fibrosis patients by active vaccination17. Passive immunization with egg yolk immunoglobulin (IgY) specific to P. aeruginosa in patients with cystic fibrosis prevented bacterial colonization and infection, perhaps by acting as an opsonin, which in turn enhanced neutrophil phagocytosis to this pathogen18–20. A recent study showed that PCN induces caspase 3-dependent human B cell (Raji Cells) death21. The aim of the present study, therefore, was to determine whether antigen-specific IgY antibodies may prevent PCN-induced Raji cell death.\n\n\nMethods\n\nPCN (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMSO at a concentration of 1 mg/ml. Five Leghorn chickens aged 3 months were subcutaneously immunized with 500 μl of Freund's complete adjuvant (Sigma-Aldrich) containing 100 μg of PCN in the back of the neck. Two weeks later, a booster was given by injecting 500 μl incomplete Freund adjuvant containing 40 μg PCN as above and the same immunization regime was repeated two weeks later. Eggs were collected one week after the last immunization and IgY was isolated by using Pierce® Chicken IgY Purification Kit (Thermo Fisher Scientific Pierche Biotechnology, Rockford, USA) according to the manufacturer. The presence of anti-PCN IgY antibodies was detected using the agarose gel precipitation test (AGPT) as previously reported22 and its concentration was assessed using the Chicken IgY ELISA Kit (Elabscience Biotechnology Co., Ltd, USA). The AGPT test was performed three times, each of 4 isolates from the first and second IgY purification results. The ELISA was then performed on two IgY batches.\n\nRaji cells, a human B cell line, obtained from central university laboratory LPPT, Universitas Gadjah Mada, Yogyakarta, Indonesia, were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 UI/ml of penicillin-streptomycin, and 250 μl/ml of amphotericin B and then incubated in 5% CO2 humidity. All materials for culture medium were purchased from Sigma-Aldrich. The cells were cultured in 96-well plates and five replicates were carried out for assays.\n\nPCN (Sigma-Aldrich) was initially dissolved in DMSO (Sigma-Aldrich) at a concentration of 1 mg/ml and then diluted in RPMI to a final concentration of 1 μg/ml, 10 μg/ml, 25 μg/ml, and 50 μg/ml. Raji cells at 2 × 104 cells incubated without the presence of PCN were used as a negative control. After exposure to various concentration of PCN then the cultures were incubated at 37°C for 24 hours. In the next experiments, the cells, at a concentration of 2 × 104 cells/well, were treated with 50 μg/ml PCN with or without the presence of various concentration (6.71 μg/ml, 13.42 μg/ml, 28.19 μg/ml, 55.49 μg/ml, 111.87 μg/ml, and 223.75 μg/ml) of anti-PCN IgY were cultured in 96-well plates and incubated for 16 hours. Cell survivability was assessed by MTT assay as described previously21. Experiments were carried out three times with 8 replicates in each group.\n\nIn order to assess cell viability, 5 × 104 cells/well were cultured on sterile coverslips in 24-well plates for 24 hours and then treated with PCN in the presence or absence of anti-PCN IgY (55.49 µg/ml) for 16 hours. Subsequently, the cells were stained with acrydine orange/ethidium bromide and viewed under Digital Carl Zeiss-Axioscope 40 (Carl Zeiss Vision, Oberkochen, Germany) by which viable and death cells appeared as green and orange/red, respectively.\n\nThe results of PCN cytotoxicity assay on Raji cells were analyzed by using one way analysis of variance followed by LSD test. Data obtained from the experiments on the effects of anti-PCN IgY on PCN-treated Raji cells was analysed by using one-way ANOVA followed by Tukey’s Test. Statistical analysis was calculated by using IBM SPSS Statistics Version 22 (SPSS Inc., IBM Corp., Chicago, IL).\n\n\nResults\n\nFollowing isolation and purification of IgY from the immunized chickens, PCN-IgY complexes were detected by using AGPT. As seen in Figure 1, clear lines of precipitates from two IgY batches in the agarose matrix indicated the presence of PCN-specific antibodies. A further assessment using ELISA demonstrated that the first batch gives high amount of specific IgY antibodies (8.95 μg/μl) than that one of the second (3.02 µg/µl) which were then used for the rest of experiments.\n\nThe presence of PCN-IgY antibody was detected through the presence of precipitates formed on agarose gel.\n\nThe results of this study showed that PCN at 1 mg/ml was toxic to the Raji cells. This cytotoxic effect of PCN on the cells was steadily increased in a dose dependent fashion (p<0.05) (Figure 2).\n\nAfter incubation with various concentration of PCN, Raji cell viabilities were assessed by MTT assay. PCN-treated Raji cell survivability was calculated against the control cells. p<0.05.\n\nFurther experimentation demonstrated that anti-PCN IgY at concentrations of 28.19 μg/mL or higher was able to suppress the cytotoxic effect of PCN on Raji cells as compared with the negative control (p<0.05) (Figure 3). No significant differences between the cells treated with PCN and specific anti-PCN IgY antibodies at the concentration above 55 μg/ml were observed, however (p>0.05) (Figure 3). Microscopically, the number of viable cells treated with PCN-IgY complexes was much higher than those treated with PCN only (Figure 4). Raw cell viability counts, along with other raw results and images, are available as Underlying data23.\n\nPCN was incubated with various concentration of IgY antibodies. Raji cells were then incubated with the PCN-IgY mixtures. Viable cells were assessed by MTT and their percentage was calculated as in Figure 2. p<0.05.\n\nRaji cells were incubated without PCN (A) and with PCN (B) or the mixture of PCN-IgY antibodies (C) and then stained with acrydine orange/ethidium bromide. Green or orange fluorescence stained cells are viable and dead cells, respectively.\n\n\nDiscussion\n\nThe results of the present study showed that PCN does induce cell death in Raji cells as also seen in our previous report21. Similarly, other also demonstrated that PCN of P. aeruginosa plays an important role in the invasion of host cells by inducing neutrophil cell death16. Therefore, efforts to inhibit excessive host cell damage induced by PCN are imminent.\n\nFurther results of the present study demonstrated that anti-PCN IgY antibodies specific to PCN significantly reduce the ability of this virulence to induce Raji cell death in a dose-dependent fashion. Whilst no previous studies showing prevention of PCN-induced cell death by antigen-specific IgY have yet been reported to our knowledge, the present results are supported by the fact that antigen-specific IgY antibodies did prevent P. aeruginosa infection in humans by both active and passive immunization17–19, suggesting that P. aeruginosa-specific IgY antibodies may inhibit cellular inflammatory responses induced by this pathogen. Antigen-specific IgY antibodies also stimulated P. aeruginosa aggregation and increased human neutrophil phagocytic activities20. The exact mechanism by which antigen-specific IgY antibodies inhibited PCN-induced Raji cell death seen in the present study remains unclear, however. Our previous study indicated that PCN induced Raji cell death via a caspase 3-activation pathway21. It seems plausible, therefore, that PCN-IgY antibody complexes may fail to activate Raji cell-derived caspase 3 and hence, inhibit cell death. However, more studies are required to delineate this speculation.\n\nThe extrapolation of the results of the present study in the therapy of P. aeruginosa infection remains to be further investigated. P. aeruginosa with its multiple mechanisms for adaptation and survival is well known as one of the main pathogens that lead to increase nosocomial infections1–4 and morbidity and mortality in cystic fibrosis patients5. The difficulty to eradicate P. aeruginosa infection has been even more complicated by the presence of MDR P. aeruginosa, and hence alternative supplemental treatment approaches have been put forward based upon its bacterial virulent factors, such as exotoxin, lipopolysaccharide, and flagellin24. Immunotherapies using IgY specific to P. aeruginosa to delay initial infection and reduce both frequency of infection and development of chronic infection seem to be promising. Both passive and active immunization with antigen-specific IgY antibodies in humans resulted in inhibition of P. aeruginosa infection. For example, oral immunization with specific IgY antibodies in patients with cystic fibrosis led to the inhibition of P. aeruginosa colonization18,19, suggesting the usefulness of antigen-specific IgY antibodies an immunotherapy to prevent P. aeruginosa infection in patients with cystic fibrosis. Therefore, PCN-specific IgY antibodies used as an immunotherapy alongside with the common antibiotic treatments for P. aeruginosa infection are highly possible.\n\nIn conclusion, the present study showed that eggs from PCN-immunized chickens contain substantial amount of IgY antibodies that recognize PCN. Furthermore, antigen-specific IgY antibodies were able to inhibit PCN-induced Raji cell death, suggesting that PCN-specific IgY antibodies may be protective against PCN-induced Raji cell death.\n\n\nData availability\n\nFigshare: Cytotoxicity of PCN.xlsx. https://doi.org/10.6084/m9.figshare.8115701.v123.\n\nThis project contains the following underlying data:\n\nCytotoxicity of PCN.xlsx (raw cell viability data following treatment with pyocyanin)\n\nThe effect of IgY on cell viability.xlsx (raw cell viability data following treatment with pyocyanin and IgY)\n\nFig 4A untreated cells.JPG (raw image used for Figure 4A)\n\nFig 4B PCN-treated cells.JPG (raw image used for Figure 4B)\n\nFig 4C PCN IgY-treated cells.JPG (raw image used for Figure 4C)\n\nIMG-AGPT.jpg (raw image of agar gel precipitation test)\n\nElisa results Sept 1.xls (raw ELISA data)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThis study was supported by a research grant (no. 1048/UN1-P.III/LT/DIT-LIT/2016) from the Ministry of Research, Technology and Higher Education, the Indonesian Government.\n\n\nAcknowledgements\n\nWe also express our gratitude to Ericko Tedy Hananto, who has helped with full responsibility during antibody preparation.\n\n\nReferences\n\nLamas Ferreiro JL, Álvarez Otero J, González González L, et al.: Pseudomonas aeruginosa urinary tract infections in hospitalized patients: Mortality and prognostic factors. PLoS One. 2017; 12(5): e0178178. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTang X, Xiao M, Zhuo C, et al.: Multi-level analysis of bacteria isolated from inpatients in respiratory departments in China. J Thorac Dis. 2018; 10(5): 2666–2675. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHattemer A, Hauser A, Diaz M, et al.: Bacterial and clinical characteristics of health care- and community-acquired bloodstream infections due to Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2013; 57(8): 3969–3975. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCaldas AR, Brandao M, Paula FS, et al.: Hypertrophic cranial pachymeningitis and skull base osteomyelitis by Pseudomonas aeruginosa: case report and review of the literature. J Clin Med Res. 2012; 4(2): 138–144. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGowan JR, Deretic V: Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev. 1996; 60(3): 539–574. PubMed Abstract \| Free Full Text\n\nJuan C, Peña C, Oliver A: Host and Pathogen Biomarkers for Severe Pseudomonas aeruginosa Infections. J Infect Dis. 2017; 215(Suppl 1): S44–S51. PubMed Abstract \| Publisher Full Text\n\nNord CE, Sjöberg L, Wadström T: Pseudomonas aeruginosa in oral infections. Acta Odontol Scand. 1972; 30(3): 371–381. PubMed Abstract \| Publisher Full Text\n\nBarnett F, Axelrod P, Tronstad L, et al.: Ciprofloxacin treatment of periapical Pseudomonas aeruginosa infection. Endod Dent Traumatol. 1988; 4(3): 132–137. PubMed Abstract \| Publisher Full Text\n\nMoradali MF, Ghods S, Rehm, BH: Pseudomonas aeruginosa Lifestyle: A Paradigm for Adaptation, Survival, and Persistence. Front Cell Infect Microbiol. 2017; 7: 39. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDietrich LE, Price-Whelan A, Petersen A, et al.: The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa. Mol Microbiol. 2006; 61(5): 1308–1321. PubMed Abstract \| Publisher Full Text\n\nBaron SS, Rowe JJ: Antibiotic action of pyocyanin. Antimicrob Agents Chemother. 1981; 20(6): 814–820. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nO’Malley YQ, Reszka KJ, Rasmussen GT, et al.: The Pseudomonas secretory product pyocyanin inhibits catalase activity in human lung epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2003; 285(5): L1077–1086. PubMed Abstract \| Publisher Full Text\n\nHall S, McDermott C, Anoopkumar-Dukie S, et al.: Cellular Effects of Pyocyanin, a Secreted Virulence Factor of Pseudomonas aeruginosa. Toxins (Basel). 2016; 8(8): pii: E236. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUlmer AJ, Pryjma J, Tarnok Z, et al.: Inhibitory and stimulatory effects of Pseudomonas aeruginosa pyocyanine on human T and B lymphocytes and human monocytes. Infect Immun. 1990; 58(3): 808–815. PubMed Abstract \| Free Full Text\n\nBianchi SM, Prince LR, McPhillips K, et al.: Impairment of apoptotic cell engulfment by pyocyanin, a toxic metabolite of Pseudomonas aeruginosa. Am J Respir Crit Care Med. 2008; 177(1): 35–43. PubMed Abstract \| Publisher Full Text\n\nManagò A, Becker KA, Carpinteiro A, et al.: Pseudomonas aeruginosa pyocyanin induces neutrophil death via mitochondrial reactive oxygen species and mitochondrial acid sphingomyelinase. Antioxid Redox Signal. 2015; 22(13): 1097–1110. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDöring G, Meisner C, Stern M, et al.: A double-blind randomized placebo-controlled phase III study of a Pseudomonas aeruginosa flagella vaccine in cystic fibrosis patients. Proc Natl Acad Sci U S A. 2007; 104(26): 11020–11025. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKollberg H, Carlander D, Olesen H, et al.: Oral administration of specific yolk antibodies (IgY) may prevent Pseudomonas aeruginosa infections in patients with cystic fibrosis: a phase I feasibility study. Pediatr Pulmonol. 2003; 35(6): 433–440. PubMed Abstract \| Publisher Full Text\n\nNilsson E, Larsson A, Olesen HV, et al.: Good effect of IgY against Pseudomonas aeruginosa infections in cystic fibrosis patients. Pediatr Pulmonol. 2008; 43(9): 892–9. PubMed Abstract \| Publisher Full Text\n\nThomsen K, Christophersen L, Bjarnsholt T, et al.: Anti-Pseudomonas aeruginosa IgY Antibodies Induce Specific Bacterial Aggregation and Internalization in Human Polymorphonuclear Neutrophils. Infect Immun. 2015; 83(7): 2686–2693. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSusilowati H, Hutomo S, Siwi DP, et al.: Caspase-3-dependent cell death in B lymphocyte caused by Pseudomonas aeruginosa pyocyanin. J Dent Indonesia. 2015; 22(2): 51–57. Publisher Full Text\n\nYork JJ, Fahey KJ: Diagnosis of infectious laryngotracheitis using a monoclonal antibody ELISA. Avian Pathol. 1988; 17(1): 173–182. PubMed Abstract \| Publisher Full Text\n\nSusilowati H: Cytotoxicity of PCN.xlsx. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8115701.v1\n\nHurley MN, Cámara M, Smyth AR: Novel approaches to the treatment of Pseudomonas aeruginosa infections in cystic fibrosis. Eur Respir J. 2012; 40(4): 1014–1023. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "50799", "date": "16 Jul 2019", "name": "Boy M. Bachtiar", "expertise": [ "Reviewer Expertise Oral microbiology", "host agent interaction" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments:\nIn this manuscript, the authors provide data account of an IgY-antibody that was purified from eggs of hens immunized with PCN secreted by P. aeruginosa. The specificity of PCN-IgY binding was determined by using AGPT, and it was further examined in vitro experiment to mimic the naturally occurring of bacterium-host cell interaction. In my opinion, it is necessary to explain (in the discussion session), when the toxin (PCN) is secreted and injected into host cells. For example, after cell contact (adherence phase).\n\nSpecific commons:\n\nMethods:\nIn the experiment, PCN was added into the cultured cell in the presence or absence of anti-PCN IgY (acted as control). This means, in the absence of the IgY, the toxin can enter the host cell (might be in adherence phase) due to the presence of a receptor, then induced the intracellular pathway leading to human B cell (Raji Cells) death (21). If this is the case, please explain it in the discussion session.\nResults:\n\nIn order to exclude chicken serum as the other IgY source, please change “the immunized chickens” to “egg of immunized chickens”.\n\nIn this study, they used a microscope (fluorescence microscope?) to compare the Raji cells number between groups tested, qualitatively, thus the obtained results was not quantitative.\n\nDiscussion:\n”anti-PCN IgY antibodies specific ………….to this virulence”. I suggest changing this virulence with this pathogen.\n\n“the present results are supported by the fact that antigen-specific IgY antibodies did prevent P. aeruginosa infection…….., suggesting that P. aeruginosa-specific IgY antibodies may inhibit cellular inflammatory responses induced by this pathogen”. Since the IgY used in this study was specifically bind to the secreted PCN, not to bacterium’s whole cell, the rationale behind using this antibody is need to be explored.\n\nAccording to the reference (16), the toxin is not having a role in the bacterial invasion into the host cell, but it involves in the mechanisms by which the bacterium kills the host cell tested (neutrophils).\n\n“Furthermore, antigen-specific IgY antibodies were……, suggesting that PCN-specific IgY antibodies may be protective against PCN-induced Raji cell death”. Not clear, what (cell surface) antigen/s that the authors assumed to bind specifically to the IgY, in addition to the secreted toxin (PCN).\n\nBased on this study, please suggest, what should be done in future studies.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "52110", "date": "27 Aug 2019", "name": "Priya Madhavan", "expertise": [ "Reviewer Expertise Immunogenetics", "Antimicrobial resistance", "Molecular Microbiology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, the study design is of an acceptable quality. Immunotherapy using IgY specific to P. aeruginosa looks promising. Minor English editing and grammar check are required.\nMagnification/Scale on Figure 4 is missing. Best to include this.\n\nWhy isn't there a positive control? I can only see results with negative control. Please provide a justification for not including a positive control.\n\nBest to also include other articles from year 2018-2019.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1008
https://f1000research.com/articles/6-748/v1	26 May 17	{ "type": "Method Article", "title": "CyTOF workflow: Differential discovery in high-throughput high-dimensional cytometry datasets", "authors": [ "Malgorzata Nowicka", "Carsten Krieg", "Lukas M. Weber", "Felix J. Hartmann", "Silvia Guglietta", "Burkhard Becher", "Mitchell P. Levesque", "Mark D. Robinson", "Malgorzata Nowicka", "Carsten Krieg", "Lukas M. Weber", "Felix J. Hartmann", "Silvia Guglietta", "Burkhard Becher", "Mitchell P. Levesque" ], "abstract": "High dimensional mass and flow cytometry (HDCyto) experiments have become a method of choice for high throughput interrogation and characterization of cell populations.Here, we present an R-based pipeline for differential analyses of HDCyto data, largely based on Bioconductor packages. We computationally define cell populations using FlowSOM clustering, and facilitate an optional but reproducible strategy for manual merging of algorithm-generated clusters. Our workflow offers different analysis paths, including association of cell type abundance with a phenotype or changes in signaling markers within specific subpopulations, or differential analyses of aggregated signals. Importantly, the differential analyses we show are based on regression frameworks where the HDCyto data is the response; thus, we are able to model arbitrary experimental designs, such as those with batch effects, paired designs and so on. In particular, we apply generalized linear mixed models to analyses of cell population abundance or cell-population-specific analyses of signaling markers, allowing overdispersion in cell count or aggregated signals across samples to be appropriately modeled. To support the formal statistical analyses, we encourage exploratory data analysis at every step, including quality control (e.g. multi-dimensional scaling plots), reporting of clustering results (dimensionality reduction, heatmaps with dendrograms) and differential analyses (e.g. plots of aggregated signals).", "keywords": [ "CyTOF", "flow cytometry", "differential analysis" ], "content": "Introduction\n\nFlow cytometry and the more recently introduced CyTOF (cytometry by time-of-flight mass spectrometry or mass cytometry) are high-throughput technologies that measure protein abundance on the surface or within cells. In flow cytometry, antibodies are labeled with fluorescent dyes and fluorescence intensity is measured using lasers and photodetectors. CyTOF utilizes antibodies tagged with metal isotopes from the lanthanide series, which have favorable chemistry and do not occur in biological systems; abundances per cell are recorded with a time-of-flight mass spectrometer. In either case, fluorescence intensities (flow cytometry) or ion counts (mass cytometry) are assumed to be proportional to the expression level of the antibody-targeted antigens of interest.\n\nDue to the differences in acquisition, further distinct characteristics should be noted. Conventional fluorophore-based flow cytometry is non-destructive and can be used to sort cells for further analysis. However, because of the spectral overlap between fluorophores, compensation of the data needs to be performed (Roederer, 2001), which also limits the number of parameters that can be measured simultaneously. Thus, standard flow cytometry experiments measure 6–12 parameters with modern systems measuring up to 20 channels (Mahnke & Roederer, 2007), while new developments (e.g. BD FACSymphony) promise to increase this capacity towards 50. Moreover, flow cytometry offers the highest throughput with tens of thousands of cells measured per second at relatively low operating costs per sample.\n\nBy using rare metal isotopes in CyTOF, cell autofluorescence can be avoided and spectral overlap is drastically reduced. However, the sensitivity of mass spectrometry results in the measurement of metal impurities and oxide formations, which need to be carefully considered in antibody panel design (e.g. through antibody concentrations and coupling of antibodies to neighboring metals). Leipold et al. recently commented that minimal spillover does not equal no spillover (Leipold, 2015). Nonetheless, CyTOF offers a high dimension of parameters measured per cell, with current panels using ~40 parameters and the promise of up to 100. Throughput of CyTOF is lower, at the rate of hundreds of cells per second, and cells are destroyed during ionization.\n\nThe ability of flow cytometry and mass cytometry to analyze individual cells at high-throughput scales has resulted in a wide range of biological and medical applications. For example, immunophenotyping assays are used to detect and quantify cell populations of interest, to uncover new cell populations and compare abundance of cell populations between different conditions, for example between patient groups (van Unen et al., 2016). Thus, it can be used as a biomarker discovery tool.\n\nVarious methodological approaches aim for biomarker discovery (Saeys et al., 2016). A common strategy, which we will refer to through this workflow as the “classic” approach, is to first identify cell populations of interest by manual gating or automated clustering (Hartmann et al., 2016; Pejoski et al., 2016). Second, using statistical tests, one can determine which of the cell subpopulations or protein markers are associated with a phenotype (e.g. clinical outcome) of interest. Typically, cell subpopulation abundance expressed as cluster cell counts or median marker expression would be used in the statistical model to relate to the sample-level phenotype.\n\nImportantly, there are many alternatives to what we propose below, and several new methods are emerging. For instance, citrus (Bruggner et al., 2014) tackles the differential discovery problem by strong over-clustering of the cells, and by building a hierarchy of clusters from very specific to general ones. Using model selection and regularization techniques, clusters and markers that associate with the outcome are identified. A new machine learning approach, CellCnn (Arvaniti & Claassen, 2016), learns the representation of clusters that are associated with the considered phenotype by means of convolutional neural networks, which makes it particularly applicable to detecting discriminating rare cell populations. However, there are tradeoffs to consider. citrus performs feature selection but does not provide significance levels, such as p-values, for the strength of associations. Due to its computational requirements, citrus can not be run on entire mass cytometry datasets and one typically must analyze a subset of the data. The “filters” from CellCnn may identify one or more cell subsets that distinguish experimental groups, while these groups may not necessarily correspond to any of the canonical cell types, since they are learned with a data-driven approach.\n\nA noticeable distinction between the machine-learning approaches and our classical regression approach is how the model is designed. citrus and CellCnn model the patient response as a function of the measured HDCyto values, whereas the classical approach models the HDCyto data itself as the response, thus putting the distributional assumptions on the experimental HDCyto data. This carries the distinct advantage that covariates (e.g. age, gender, batch) can be included, together with finding associations of the phenotype to the predictors of interest (e.g. cell type abundance). Specifically, neither citrus nor CellCnn are able to directly account for complex designs, such as paired experiments or presence of batches.\n\nWithin the classical approach, hybrid methods are certainly possible, where discovery of interesting cell populations is done with one algorithm, and quantifications or signal aggregations are modeled in standard regression frameworks. For instance, CellCnn provides p-values from a t-test or Mann-Whitney U-test conducted on the frequencies of previously detected cell populations. The models we propose below are flexible extensions of this strategy.\n\nStep by step, this workflow presents differential discovery analyses assembled from a suite of tools and methods that, in our view, lead to a higher level of flexibility and robust, statistically-supported and interpretable results. Cell population identification is conducted by means of unsupervised clustering using the FlowSOM and ConsensusClusterPlus packages, which together were among the best performing clustering approaches for high-dimensional cytometry data (Weber & Robinson, 2016). Notably, FlowSOM scales easily to millions of cells and thus no subsetting of the data is required.\n\nTo be able to analyze arbitrary experimental designs (e.g. batch effects, paired experiments, etc.), we show how to conduct the differential analysis of cell population abundances using the generalized linear mixed models (GLMM) and of marker intensities using linear models (LM) and linear mixed models (LMM). Model fitting is performed with lme4 and stats packages, and hypothesis testing with the multcomp package.\n\nWe use the ggplot2 package as our graphical engine. Notably, we propose a suite of useful visual representations of HDCyto data characteristics, such as an MDS (multidimensional scaling) plot of aggregated signal for exploring sample similarities. The obtained cell populations are visualized using dimension reduction techniques (e.g. t-SNE via the Rtsne package) and heatmaps (via the pheatmap package) to represent characteristics of the annotated cell populations and identified biomarkers.\n\nThe workflow is intentionally not fully automatic. First, we strongly advocate for exploratory data analysis to get an understanding of data characteristics before formal statistical modeling. Second, the workflow involves an optional step where the user can manually merge and annotate clusters (see Cluster merging and annotation section) but in a way that is easily reproducible. The CyTOF data used here (see Data description section) is already preprocessed; i.e. the normalization and de-barcoding, as well as removal of doublets, debris and dead cells, were already performed. To see how such an analysis could be performed, please see the Data preprocessing section.\n\nNotably, this workflow is equally applicable to flow or mass cytometry datasets, for which the preprocessing steps have already been performed. In addition, the workflow is modular and can be adapted as new algorithms or new knowledge about how to best use existing tools comes to light. Alternative clustering algorithms such as the popular PhenoGraph algorithm (Levine et al., 2015) (e.g. via the Rphenograph package), dimensionality reduction techniques, such as diffusion maps (Haghverdi et al., 2015) via the destiny package (Angerer et al., 2016)), and SIMLR (Wang et al., 2017) via the SIMLR package could be inserted to the workflow.\n\n\nData description\n\nWe use a subset of CyTOF data originating from Bodenmiller et al. (Bodenmiller et al., 2012) that was also used in the citrus paper (Bruggner et al., 2014). Specifically, we perform our analysis on samples of peripheral blood mononuclear cells (PBMCs) from 8 healthy donors, where for each individual, an unstimulated and a stimulated sample (for 30 minutes with B cell receptor/Fc receptor crosslinking, known as BCR/FcR-XL) was collected, resulting in 16 samples in total. For each sample, 14 signaling markers and 10 cell surface markers were measured.\n\nThe original data is available from the Cytobank report. The subset used here can be downloaded from the Citrus Cytobank repository (files with _BCR-XL.fcs or _Reference.fcs endings) or from our web server (see the Data import section).\n\nIn both the Bodenmiller et al. and citrus manuscripts, the 10 lineage markers were used to identify cell subpopulations. These were then investigated for differences between reference and stimulated cell subpopulations separately for each of the 14 functional markers. The same strategy is used in this workflow; 10 lineage markers are used for cell clustering and 14 functional markers are tested for differential expression between the reference and BCR/FcR-XL stimulation. Even though differential analysis of cell abundance was not in the scope of the Bodenmiller et al. experiment, we present it here to highlight the generality of the discovery.\n\n\nData preprocessing\n\nConventional flow cytometers and mass cytometers produce .fcs files that can be manually analyzed using programs such as FlowJo [TriStar] or Cytobank (Kotecha et al., 2010), or using the R/Bioconductor packages, such as the flowCore package (Ellis et al., 2017). During this initial analysis step, dead cells are removed, compensation is checked and with simple two dimensional scatter plots (e.g. marker intensity versus time), marker expression patterns are checked. Often, modern experiments are barcoded in order to remove analytical biases due to individual sample variation or acquisition time. Preprocessing steps including normalization using bead standards, de-barcoding and compensation can be completed with the CATALYST package, which provides an implementation of the de-barcoding algorithm described by Zunder et al. (Zunder et al., 2015) and the bead-based normalization from Finck et al. (Finck et al., 2013). Of course, preprocessing steps can occur using custom scripts within R or outside of R (e.g. Normalizer (Finck et al., 2013)).\n\n\nData import\n\nWe recommend as standard practice to keep an independent record of all samples collected, with additional information about the experimental condition, including sample or patient identifiers, processing batch and so on. That is, we recommend having a trail of metadata for each experiment. In our example, the metadata file, PBMC8_metadata.xlsx, can be downloaded from the Robinson Lab server with the download.file function. For the workflow, the user should place it in the current working directory (getwd()). Here, we load it into R with the read_excel function from the readxl package and save it into a variable called md, but other file types and interfaces to read them in are also possible.\n\nThe data frame md contains the following columns:\n\nfile_name with names of the .fcs files corresponding to the reference (suffix “Reference”) and BCR/FcR-XL stimulation (suffix “BCR-XL”) samples,\n\nsample_id with shorter unique names for each sample containing information about conditions and patient IDs,\n\ncondition describes whether samples originate from the reference (Ref) or stimulated (BCRXL) condition,\n\npatient_id defines the IDs of patients.\n\nThe sample_id variable is used as row names in metadata and will be used all over the workflow to label the samples. It is important to carefully check whether variables are of the desired type (factor, numeric, character), since input methods may convert columns into different data types. For the statistical modeling, we want to make the condition variable a factor with the reference (Ref) samples being the reference level, where the order of factor levels can be defined with the levels parameter of the factor function. We also specify colors for the different conditions in a variable color_conditions.\n\n\n\nThe .fcs files listed in the metadata can be downloaded manually from the Citrus Cytobank repository or automatically from the Robinson Lab server where they are saved in a compressed archive file, PBMC8_fcs_files.zip.\n\n\n\nTo load the content of the .fcs files into R, we use the flowCore. Using read.flowSet, we read in all files into a flowSet object, which is a general container for HDCyto data. Importantly, read.flowSet and the underlying read.FCS functions, by default, may transform the marker intensities and remove cells with extreme positive values. We keep these options off to be sure that we control the exact preprocessing steps.\n\n\n\nIn our example, information about the panel is also available in a file called PBMC8_panel.xlsx, and can be downloaded from the Robinson Lab server and loaded into a variable called panel. It contains columns for Isotope and Metal that define the atomic mass number and the symbol of the chemical element conjugated to the antibody, respectively, and Antigen, which specifies the protein marker that was targeted; two additional columns specify whether a channel belongs to the lineage or surface type of marker.\n\nThe isotope, metal and antigen information that the instrument receives is also stored in the flowFrame (container for one sample) or flowSet (container for multiple samples) objects. You can type fcs_raw[[1]] to see the first flowFrame, which contains a table with columns name and desc. Their content can be accessed with functions pData(parameters()), which is identical for all the flowFrame objects in the flowSet. The variable name corresponds to the column names in the flowSet object, you can type in R colnames(fcs_raw).\n\nIt should be checked that elements from panel can be matched to their corresponding entries in the flowSet object to make the analysis less prone to subsetting mistakes. Here, for example, the entries in panel$Antigen have their exact equivalents in the desc columns of the flowFrame objects. In the following analysis, we will often use marker IDs as column names in the tables containing expression values. As a cautionary note, during object conversion from one type to another (e.g. in the creation of data.frame from a matrix), some characters (e.g. dashes) in the dimension names are replaced with dots, which may cause problems in matching. To avoid this problem, we replace all the dashes with underscores. Also, we define two variables that indicate the lineage and functional markers.\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\nData transformation\n\nUsually, the raw marker intensities read by a cytometer have strongly skewed distributions with varying ranges of expression, thus making it difficult to distinguish between the negative and positive cell populations. It is common practice to transform CyTOF marker intensities using, for example, arcsinh (hyperbolic inverse sine) with cofactor 5 (Bendall et al., 2011 Figure S2; Bruggner et al., 2014) to make the distributions more symmetric and to map them to a comparable range of expression, which is important for clustering. A cofactor of 150 has been promoted for flow cytometry, but users are free to implement alternative transformations, some of which are available from the transform function of the flowCore package. In the following step, we include only those channels that correspond to the lineage and functional markers. We also rename the columns in the flowSet to the antigen names from panel$desc.\n\n\n\nFor some of the further analysis, it is more convenient for us to work using a matrix (called expr) that contains marker expression for cells from all samples. We create such a matrix with the fsApply function that extracts the expression matrices (function exprs) from each element of the flowSet object.\n\n\n\nAs the ranges of marker intensities can vary substantially, we apply another transformation that scales expression of all markers to values between 0 and 1 using low (e.g. 1%) and high (e.g. 99%) percentiles as the boundary. This additional transformation of the arcsinh-transformed data can sometimes give better representation of relative differences in marker expression between annotated cell populations, however, it is only used here for visualization.\n\n\n\n\nDiagnostic plots\n\nWe propose some quick checks to verify whether the data we analyze globally represents what we expect; for example, whether samples that are replicates of one condition are more similar and are distinct from samples from another condition. Another important check is to verify that marker expression distributions do not have any abnormalities such as having different ranges or distinct distributions for a subset of the samples. This could highlight problems with the sample collection or HDCyto acquisition, or batch effects that were unexpected. Depending on the situation, one can then consider removing problematic markers or samples from further analysis; in the case of batch effects, a covariate column could be added to the metadata table and used below in the statistical analyses.\n\nThe step below generates a plot with per-sample marker expression distributions, colored by condition (see Figure 1). Here, we can already see distinguishing markers, such as pNFkB and CD20, between stimulated and unstimulated conditions.\n\nTwo conditions: unstimulated (Ref) and stimulated with BCR/FcR-XL (BCRXL) for each of the 8 healthy donors are presented and colored by experimental condition.\n\n\n\nAnother spot check is the number of cells per sample (see Figure 2). This plot can be used as a guide together with other readouts to identify samples where not enough cells were assayed.\n\nBars are colored by experimental condition: unstimulated (Ref) and stimulated with BCR/FcR-XL (BCRXL). Numbers in the names on the x-axis indicate patient IDs. Numbers on top of the bars indicate the cell counts.\n\n\n\nIn transcriptomics applications, one of the most utilized exploratory plots is the multi-dimensional scaling (MDS) plot or a principal component analysis (PCA) plot. Such plots show similarities between samples measured in an unsupervised way and give a sense of how much differential expression can be detected before conducting any formal tests. An MDS plot can be generated with the plotMDS function from the limma package. In transcriptomics, distances between samples are calculated based on the expression of the top varying genes. We propose a similar plot for HDCyto data using median marker expression over all cells to calculate dissimilarities between samples (other aggregations are also possible, and one could reduce the number of top varying markers to include in the calculation). Ideally, samples should cluster well within the same condition, although this depends on the magnitude of the difference between experimental conditions. With this diagnostic, one can identify the outlier samples and eliminate them if the circumstances warrant it.\n\nIn our MDS plot on median marker expression values (see Figure 3), we can see that the first dimension (MDS1) separates the unstimulated and stimulated samples reasonably well. The second dimension (MDS2) represents, to some degree, differences between patients. Most of the samples that originate from the same patient are placed at a similar point along the y-axis, for example, samples from patients 7, 5, and 8 are at the top of the plot, samples from patient 4 are located at the bottom of the plot. This also indicates that the marker expression of individual patients is driving similarity and perhaps should be formally accounted for in the downstream statistical modeling.\n\n\n\nCalculations are based on the median (arcsinh-transformed) marker expression of 10 lineage markers and 14 functional markers across all cells measured for each sample. Distances between samples on the plot approximate the typical change in medians. Numbers in the label names indicate patient IDs.\n\nIn contrast to genomic applications, the number of variables measured for each sample is much lower in HDCyto data. In the former, thousands of genes are surveyed, whereas in the latter, ~20-50 antigens are typically targeted. Similar to the MDS plot above, a heatmap of the same data also gives insight into the structure of the data. The heatmap shows median marker intensities with clustered columns (samples) and rows (markers). We have used hierarchical clustering with average linkage and euclidean distance, but also Ward’s linkage could be used (Bruggner et al., 2014), and in CyTOF applications, a cosine distance shows good performance (Bendall et al., 2014). In this plot, we can see which markers drive the observed clustering of samples (see Figure 4).\n\nThe two conditions: unstimulated (Ref) and stimulated with BCR/FcR-XL (BCRXL) for each of the 8 healthy donors are presented and colored by experimental condition. Dendrograms are based on hierarchical clustering with Euclidean distance metric and average linkage. Numbers in the column label names indicate patient IDs.\n\nAs with the MDS plot, the dendrogram separates the reference and stimulated samples very well. Also, similar groupings of patients within experimental conditions are observed (patients 1-2, 5-7-8 and 3-4 are together in both conditions).\n\n\n\n\nMarker ranking based on the non-redundancy score\n\nIn this step, we identify the ability of markers to explain the variance observed in each sample. In particular, we calculate the PCA-based non-redundancy score (NRS) from Levine et al. (Levine et al., 2015). Markers with higher score explain a larger portion of variability present in a given sample.\n\nThe average NRS can be used to select a subset of markers that are non-redundant in each sample but at the same time capture the overall diversity between samples. Such a subset of markers can be then used for cell population identification analysis (i.e. clustering). We note that there is no precise rule on how to choose the right cutoff for marker inclusion, but one of the approaches is to select a suitable number of the top-scoring markers. The number can be chosen by analyzing the plot with the NR scores (see Figure 5), where the markers are sorted by the decreasing average NRS. One can drop out markers that are not likely to distinguish cell populations of interest, even if they have high scores, and add in markers with low scores but known to be important in discerning cell subgroups (Levine et al., 2015).\n\nThe points represent the per-sample NR scores (colored by experimental conditions), while open white circles indicate the mean NR scores from all the samples. Markers on the x-axis are sorted according to the decreasing average NRS.\n\nIn the dataset considered here (Bodenmiller et al., 2012; Bruggner et al., 2014) we want to use all the 10 lineage markers, so that there is no explicit need to restrict the set of cell surface markers. There may be other situations where this feature selection step would be of interest.\n\n\n\n\nCell population identification with FlowSOM and ConsensusClusterPlus\n\nCell population identification typically has been carried out by manual gating, a method based on visual inspection of a series of two-dimensional scatterplots. At each step, a subset of cells, either positive or negative for the two visualized markers, is selected and further stratified in the subsequent iterations until populations of interest across a range of marker combinations are captured. However, manual gating has drawbacks, such as subjectivity, bias toward well-known cell types, and inefficiency when analyzing large datasets, which also contribute to a lack of reproducibility (Saeys et al., 2016).\n\nConsiderable effort has been made to improve and automate cell population identification, such as unsupervised clustering (Aghaeepour et al., 2013). However, not all methods scale well in terms of performance and speed from the lower dimensionality flow cytometry data to the higher dimensionality mass cytometry data (Weber & Robinson, 2016), since clustering in higher dimensions can suffer the “curse of dimensionality”.\n\nBeside the mathematical and algorithmic challenges of clustering, cell population identification may be difficult due to the chemical and biological aspects of the cytometry experiment itself. Therefore, caution should be taken when designing panels aimed at detecting rare cell populations by assigning higher sensitivity metals to rare markers. The right choice of a marker panel used for clustering can also be important. It should include all markers that are relevant for cell type identification.\n\nIn this workflow, we conduct cell clustering with FlowSOM (Van Gassen et al., 2015) and ConsensusClusterPlus (Wilkerson & Hayes, 2010), which appeared amongst the fastest and best performing clustering approaches in a recent study of HDCyto datasets (Weber & Robinson, 2016). This ensemble showed strong performance in detecting both high and low frequency cell populations and is one of the fastest methods to run, which enables its interactive usage. We use a slight modification of the original workflow presented in the FlowSOM vignette, which we find more flexible. In particular, we directly call the ConsensusClusterPlus function that is embedded in metaClustering_consensus. Thus, we are able to access all the functionality of the ConsensusClusterPlus package to identify the number of clusters.\n\nThe FlowSOM workflow consists of three main steps. First, a self-organizing map (SOM) is built using the BuildSOM function, where cells are assigned according to their similarities to 100 (by default) grid points (or, so-called codebook vectors or codes) of the SOM. The building of a minimal spanning tree, which is mainly used for graphical representation of the clusters, is skipped in this pipeline. And finally, metaclustering of the SOM codes, is performed directly with the ConsensusClusterPlus function. Additionally, we add an optional round of manual expert-based merging of the metaclusters and allow this to be done in a reproducible fashion.\n\nFlowSOM output can be sensitive to random starts (Weber & Robinson, 2016). To make results reproducible, one must specify the seed for the random number generation in R using function set.seed. It is also advisable to rerun analyses with multiple random seeds, for two reasons. First, one can see how robust the detected clusters are, and second, when the goal is to find smaller cell populations, it may happen that, in some runs, random starting points do not represent rare cell populations, as the chance of selecting starting cells from them is low and they are merged into a larger cluster.\n\nIt is important to point out that we cluster all cells from all samples together. This strategy is beneficial, since we label cell populations only once and the mapping of cell types between samples is automatically consistent. In our analysis, cell populations are identified using only the 10 lineage markers as defined in the BuildSOM function with the colsToUse argument.\n\n\n\nAutomatic approaches for selecting the number of clusters in HDCyto data do not always succeed (Weber & Robinson, 2016). In general, we therefore recommend some level of over-clustering, and if desired, manual merging of clusters. Such a hierarchical approach is especially suited when the goal is to detect smaller cell populations.\n\nThe SPADE analysis performed by Bodenmiller et al. (Bodenmiller et al., 2012) identified 6 main cell types (T-cells, monocytes, dendritic cells, B-cells, NK cells and surface- cells) that were further stratified into 14 more specific subpopulations (CD4+ T-cells, CD8+ T-cells, CD14+ HLA-DR high monocytes, CD14+ HLA-DR med monocytes, CD14+ HLA-DR low monocytes, CD14- HLA-DR high monocytes, CD14- HLA-DR med monocytes, CD14- HLA-DR low monocytes, dendritic cells, IgM+ B-cells, IgM-B-cells, NK cells, surface- CD14+ cells and surface-CD14- cells). In our analysis, we are interested in identifying the 6 main PBMC populations, including: CD4+ T-cells, CD8+ T-cells, monocytes, dendritic cells, NK cells and B-cells. Following the concept of over-clustering we perform the metaclustering of the (by default) 100 SOM codes into more than expected number of groups. For example, stratification into 20 groups should give enough resolution. We can explore the clustering in a wide variety of visualizations: t-SNE plots, heatmaps and a plot generated by ConsensusClusterPlus called “delta area”.\n\nWe call ConsensusClusterPlus with maximum number of clusters maxK = 20 and other clustering parameters set to the values as in the metaClustering_consensus function. Again, to ensure that the analyses are reproducible, we define the random seed.\n\n\n\nWe can then investigate characteristics of identified clusters with heatmaps that illustrate median marker expression in each cluster (see Figure 6). As the range of marker expression can vary substantially from marker to marker, we use the 0-1 transformed data for some visualizations. However, to stay consistent with FlowSOM and ConsensusClusterPlus, we use the (arcsinh-transformed) unscaled data to generate the dendrogram of the hierarchical structure of metaclusters.\n\nThe color in the heatmap represents the median of the arcsinh, 0-1 transformed marker expression. The dendrogram on the left represents the hierarchical similarity between the 20 metaclusters (metric: Euclidean distance; linkage: average). Values in the brackets indicate the relative size of a given cluster.\n\nInstead of using only medians, which do not give a full representation of cluster specifics, one can plot the entire marker expression distribution in each cluster (see Figure 7). Such a plot gives more detailed profile of each cluster, but represents an increase in the amount of information to interpret. Heatmaps give the overall overview of clusters, are quicker and easier to interpret, and together with the dendrogram can be a good basis for further cluster merging (see Cluster merging and annotation section).\n\nBlue densities represent marker expression for cells in the given cluster. Grey densities are calculated from all the cells and serve as a reference.\n\nSince we will use the heatmap and density plots again later on in this workflow, in code chunks below, we create wrapper functions that generate these two types of plots.\n\n\n\n\n\n\n\n\n\n\n\n\n\nOne of the most popular plots for representing single cell data are t-SNE plots, where each cell is represented in a lower, usually two-dimensional, space computed using t-stochastic neighbor embedding (t-SNE) (Van Der Maaten & Hinton, 2008). More generally, dimensionality reduction techniques represent the similarity of points in 2 or 3 dimensions, such that similar objects in high dimensional space are also similar in lower dimensional space. Mathematically, there are a myriad of ways to define this similarity. For example, principal components analysis (PCA) uses linear combinations of the original features to find orthogonal dimensions that show the highest levels of variability; the top 2 or 3 principal components can then be visualized.\n\nNevertheless, there are few notes of caution when using t-SNE or any other dimensionality reduction technique. Since they are based on preserving similarities between cells, those that are similar in the original space will be close in the 2D/3D representation, but the opposite does not always hold. In our experience, t-SNE with default parameters for HDCyto data is often suitable; for more guidance on the specifics of t-SNE, see How to Use t-SNE Effectively (Wattenberg et al., 2016). Due to the stochastic nature of t-SNE optimization, rerunning the method will result in different lower dimensional projections, thus it is advisable to run it a few times to identify the common trends and get a feeling about the variability of the results. As with other methods, to be sure that the analysis is reproducible, the user can define the random seed.\n\nt-SNE is a method that requires significant computational time to process the data even for tens of thousands of cells. CyTOF datasets are usually much larger and thus to keep running times reasonable, one may use a subset of cells; for example, here we use 2000 cells from each sample. The t-SNE map below is colored according to the expression level of the CD4 marker, highlighting that the CD4+ T-cells are placed to the left side of the plot (see Figure 8). In this way, one can use a collection of markers to highlight where cell types of interest are located on the map.\n\nFrom each of the 16 samples, 2000 cells were randomly selected. Cells are colored according to the expression level of the CD4 marker.\n\nInstead of t-SNE, one could also use other dimension reduction techniques, such as PCA, diffusion maps, SIMLR (Wang et al., 2017) or isomaps, some of which are conveniently available via the cytof_dimReduction function from the cytofkit package (Chen et al., 2016). To speed up the t-SNE analysis, one could use a multicore version that is available via the Rtsne.multicore package. Alternative algorithms, such as largeVis (Tang et al., 2016) (available via the largeVis package), can be used for dimensionality reduction of very large datasets without downsampling. Alternatively, the dimensionality reduction can be performed on the codes of the SOM, at a resolution specified by the user (see Figure 12).\n\n\n\n\n\n\n\n\n\nWe can color the cells by cluster. Ideally, cells of the same color should be close to each other (see Figure 9). When the plots are further stratified by sample (see Figure 10), we can verify whether similar cell populations are present in all replicates, which can help in identifying outlying samples. Optionally, stratification can be done by condition (see Figure 11). With such a spot-check plot, we can inspect whether differences in cell abundance are strong between conditions, and we can identify distinguishing clusters.\n\nFrom each of the 16 samples, 2000 cells were randomly selected. Cells are colored according to the 20 cell populations obtained with FlowSOM after the metaclustering step with ConsensusClusterPlus.\n\n\n\n\n\n\n\nThe SOM codes represent characteristics of the 100 (by default) clusters generated in the first step of the FlowSOM pipeline. Their visualization can also be helpful in understanding the cell population structure and determining the number of clusters. Ultimately, the metaclustering step uses the codes and not the original cells. We treat the codes as new representative cells and apply the t-SNE dimension reduction to visualize them in 2D (see Figure 12). The size of the points corresponds to the number of cells that were assigned to a given code. The points are colored according to the results of metaclustering. Since we have only 100 data points, the t-SNE analysis is fast.\n\n(A) t-SNE plot and (B) PCA plot representing the 100 SOM codes in the PBMC dataset colored according to the metaclustering with ConsensusClusterPlus into 20 cell populations. The SOM codes represent characteristics of the 100 (by default) clusters generated in the first step of the FlowSOM pipeline. The size of the points corresponds to the number of cells that were assigned to a given code.\n\nAs there are multiple ways to mathematically define similarity in high dimensional space, it is always good practice visualizing projections from other methods to see how consistent the observed patterns are. For instance, we also represent the FlowSOM codes via the first two principal components (see Figure 12).\n\n\n\n\n\n\n\n\n\nIn our experience, manual merging of clusters leads to slightly different results compared to an algorithm with a specified number of clusters. In order to detect somewhat rare populations, some level of over-clustering is necessary so that the more subtle populations become separated from the main populations. In addition, merging can always follow an over-clustering step, but splitting of existing clusters is not generally feasible.\n\nIn our setup, over-clustering is also useful when the interest is identifying the “natural” number of clusters present in the data. In addition to the t-SNE plots, one could investigate the delta area plot from the ConsensusClusterPlus package and the hierarchical clustering dendrogram of the over-clustered subpopulations, as shown below.\n\nIn our example, we expect around 6 specific cell types, and we have performed FlowSOM clustering into 20 groups as a reasonable over-estimate. After analyzing the heatmaps and t-SNE plots, we can clearly see that stratification of the data into 20 clusters may be too strong. Many clusters are placed very close to each other, indicating that they could be merged together. The same can be deduced from the heatmaps, highlighting that marker expression patterns for some neighboring clusters are very similar. Cluster merging and annotating is somewhat manual, based partially on visual inspection of t-SNE plots and heatmaps and thus, benefits from expert knowledge of the cell types.\n\nIn our experience, the main reference for manual merging of clusters is the heatmap of marker characteristics across metaclusters, with dendrograms showing the hierarchy of similarities. Such plots aggregate information over many cells and thus show average marker expression for each cluster. Together with dimensionality reduction, these plots give good insight into the relationships between clusters and the marker levels within each cluster. Given expert knowledge of the cell types and markers, it is then left to the researcher to decide how exactly to merge clusters (e.g., with higher weight given to some markers).\n\nThe dendrogram highlights the similarity between the metaclusters and can be used explicitly for the merging. However, there are reasons why we would not always follow the dendrogram exactly. In general, when it comes to clustering, blindly following the hierarchy of codes will lead to identification of populations of similar cells, but it does not necessarily mean that they are of biological interest. The distances between metaclusters are calculated across all the markers, and it may be that some markers carry higher weight for certain cell types. In addition, different linkage methods may lead to different hierarchy, especially when clusters are not fully distinct. Another aspect to consider in cluster merging is the cluster size, represented in the parentheses next to the cluster label in our plots. If the cluster size is very small, but the cluster seems relevant and distinct, one can keep it as separate. However, if it is small and different from the neighboring clustering only in a somewhat unimportant marker, it could be merged. And, if some of the metaclusters do not represent any specific cell types, they could be dropped out of the downstream analysis instead of being merged. However, in case an automated solution for cluster merging is truly needed, one could use the cutree() function applied to the dendrogram.\n\nBased on the seed that was set, cluster merging of the 20 metaclusters is defined in the PBMC8_cluster_merging1.xlsx file on the Robinson Lab server with the IDs of the original clusters and new cluster names, and we save it as a cluster_merging1 data frame. The expert has annotated 8 cell populations: CD8 T-cells, CD4 T-cells, B-cells IgM-, B-cells IgM+, NK cells, dendritic cells (DCs), monocytes and surface negative cells; monocytes could be further subdivided based on HLA-DR into high, medium and low subtypes.\n\n\n\n\n\n\n\nWe update the t-SNE plot with the new annotated cell populations, Figure 13.\n\nFrom each of the 16 samples, 2000 cells were randomly selected. Cells are colored according to the manual merging of the 20 cell populations obtained with FlowSOM into 8 PBMC populations.\n\n\n\nOne of the usefull representations of merging is a heatmap of median marker expression in each of the original clusters, which are labeled according to the proposed merging, Figure 14.\n\n\n\nTo get a final summary of the annotated cell types, one can plot a heatmap of median marker expression, calculated based on the cells in each of the annotated populations, Figure 15.\n\nThe heat represents the median of arcsinh and 0-1 transformed marker expression. Values in the brackets indicate the relative size of each of the cell populations across all the samples.\n\nThe ConsensusClusterPlus package provides visualizations that can help to understand the metaclustering process and the characteristics of the analyzed data. For example, the delta area plot (see Figure 16) highlights the amount of extra cluster stability gained when clustering into k groups as compared to k-1 groups (from k=2 to k=20). It can be expected that high stability of clusters can be reached when clustering into the number of groups that best fits the data. Thus, using the delta area plot could help finding the “natural” number of clusters present in the data, which would correspond to the value of k where there is no appreciable increase in stability. This strategy can be referred as the “elbow criterion”. For more details about the meaning of this plot, the user can refer to the original description of the consensus clustering method (Monti et al., 2003).\n\nThe delta area score (y-axis) indicates the relative increase in cluster stability obtained when clustering the 100 SOM codes generated by FlowSOM into k groups (x-axis).\n\nThe elbow criterion is quite subjective since the “appreciable” increase is not defined exactly. For example, in the delta plot below, we could say that the last point before plateau is for k=6, or for k=5, or for k=3, depending on our perception of sufficient decrease of the delta area score. Moreover, the exact point where a plateau is reached may vary for runs with different random seeds, the drop may not always be so sharp and and the function is not guaranteed to be decreasing. It is advisable to investigate more of those plots and the resulting t-SNE and heatmaps before drawing any conclusions about the final number of “natural” clusters.\n\nManual merging of up to 20 clusters can be laborious. To simplify this task, one could reduce the strength of over-clustering and allow the metaclustering method to do a part of the merging, which then can be completed manually. Analyzing the delta plot from the right side, we can see how much we can reduce the strength of over-clustering while still obtaining stable clusters. In parallel, one should check the heatmaps to see whether the less stringent stratification is able to capture cell populations of interest.\n\n\n\nAs an example, we chose to reduce the strength of metaclustering to 12 groups.\n\n\n\nIn the t-SNE plot (see Figure 17), we can see that many small clusters obtained when stratifying data into 20 groups are now merged together, which should simplify the new cluster annotation.\n\nFrom each of the 16 samples, 2000 cells were randomly selected. Cells are colored according to the 12 cell populations obtained with FlowSOM after the metaclustering step with ConsensusClusterPlus.\n\n\n\n\n\nOver-clustering into as few as 12 groups still allows us to identify the same 8 cell populations as when merging 20 clusters (see Figure 18–Figure 21), but it is simpler to define since fewer profiles need to be manually scanned. The expert-based merging is saved in the PBMC8_cluster_merging2.xlsx file on the Robinson Lab server.\n\nThe heat represents the median of arcsinh and 0-1 transformed marker expression. The dendrogram on the left represents the hierarchical similarity between the 12 metaclusters (metric: Euclidean distance; linkage: average). Values in the brackets indicate the relative size of a given cluster.\n\nFrom each of the 16 samples, 2000 cells were randomly selected. Cells are colored according to the 12 metaclusters obtained with FlowSOM after the metaclustering step with ConsensusClusterPlus.\n\nThe heat represents the median of arcsinh and 0-1 transformed marker expression. Values in the brackets indicate the relative size of each of the cell populations across all the samples.\n\n\n\n\n\n\n\n\n\n\n\n\n\nThe manual merging of 20 (or 12) clusters by an expert resulted in identification of 8 cell populations. To highlight the impact of manual merging versus algorithm-defined subpopulations, we compare to the results of an automated cluster merging that is set to stratify the data also into 8 clusters. We extract the result from the ConsensusClusterPlus output. Out of interest, we can see which clusters are split by tabulating the cell labels.\n\n\n\n\n\n\n\nIn the t-SNE map (see Figure 22), we can see that part of the new cell populations (cluster 7, 1 and 4, 2, 5 and 8) overlap substantially with populations obtained by the means of manual merging (CD4 T-cells, B-cells, surface-, monocytes and DC). However, cells that belong to clusters 3 and 6 are subdivided in a different manner according to the manual merging. Cluster 3 consists of CD8 T-cells and NK cells, and the latter cannot be identified anymore based on the heatmap corresponding to clustering into 8 groups (see Figure 23).\n\n\n\n\n\nt-SNE plot with cells colored according to (A) the expert merging of 12 metaclusters obtained with FlowSOM into 8 PBMC populations; and (B) the 8 automatically detected with FlowSOM metaclusters.\n\nThe heat represents the median of arcsinh and 0-1 transformed marker expression. The dendrogram on the left represents the hierarchical similarity between the 8 metaclusters (metric: Euclidean distance; linkage: average). Values in the brackets indicate the relative size of a given cluster.\n\nThe example above highlights the difference between automatic clustering and manual merging of algorithm-generated clusters in the search for biologically meaningful cell populations. Automated and manual merging may give different weight to marker importance and thus result in different populations being detected. However, in our view, the manual merging done here in a reproducible fashion results in a more biologically meaningful cell stratification.\n\n\nDifferential analysis\n\nFor the dataset used in this workflow (Bodenmiller et al., 2012; Bruggner et al., 2014), we perform three types of analyses that aim to identify subsets of PBMCs and signaling markers that respond to BCR/FcR-XL stimulation, by comparing stimulated samples to unstimulated samples. We first describe the differential abundance of the defined cell populations, followed by differential analysis of marker expression within each cluster. Finally, differential analysis of the overall aggregated marker expression could also be of interest.\n\nThe PBMC subset analyzed in this workflow originates from a paired experiment, where samples from 8 patients were treated with 12 different stimulation conditions for 30 minutes, together with unstimulated reference samples (Bodenmiller et al., 2012). This is a natural example where one would choose a mixed model to model the response (abundance or marker signal), and patients would be treated as a random effect. In this way, one can formally account for within-patient variability, observed to be quite strong in the MDS plot (see MDS plot section), and this should give a gain in power to detect differences between conditions.\n\nWe use the stats and lme4 packages to fit the fixed and mixed models, respectively, and the multcomp package for hypothesis testing. In all differential analyses here, we want to test for differences between the reference (Ref) and BCR/FcR-XL treatment (BCRXL). The fixed model formula is straightforward: ∼ condition, where condition indicates the treatment group. The corresponding full model design matrix consists of the intercept and dummy variable indicating the treated samples. In the presence of batches, one can include them in the model by using a formula ∼ condition + batch, or if they affect the treatment, a formula with interactions ∼ condition * batch.\n\nFor testing, we use the general linear hypotheses function glht, which allows testing of arbitrary hypotheses. The linfct parameter specifies the linear hypotheses to be tested. It should be a matrix where each row corresponds to one comparison (contrast), and the number of columns must be the same as in the design matrix. In our analysis, the contrast matrix indicates that the regression coefficient corresponding to conditionBCRXL is tested to be equal to zero; i.e. we test the null hypothesis that there is no effect of the BCR/FcR-XL treatment. The result of the test is a p-value, which indicates the probability of observing an as strong (or stronger) difference between the two conditions assuming the null hypothesis is true.\n\nTesting is performed on each cluster and marker separately, resulting in 8 tests for differential abundance (one for each merged population), 14 tests for overall differential marker expression analysis and 8 ´ 14 tests for differential marker expression within-populations. Thus, to account for the multiple testing correction, we apply the Benjamini & Hochberg adjustment to each of them using an FDR cutoff of 5%.\n\n\n\n\n\n\n\n\n\n\n\nDifferential analysis of cell population abundance compares the proportions of cell types across experimental conditions and aims to highlight populations that are present at different ratios. First, we calculate two tables: one that contains cell counts for each sample and population and one with the corresponding proportions of cell types by sample. The proportions are used only for plotting, since the statistical modeling takes the cell counts by cluster and sample as input.\n\n\n\n\n\nFor each sample, we plot its PBMC cell type composition represented with colored bars, where the size of a given stripe reflects the proportion of the corresponding cell type in a given sample (see Figure 24).\n\n\n\nThe 8 cell populations are a result of manual merging of the 20 FlowSOM metaclusters.\n\nIt may be quite hard to see the differences in cluster abundances in the plot above, especially for clusters with very low frequency. And, since boxplots cannot represent multimodal distributions, we show boxplots with jittered points of the sample-level cluster proportions overlaid (see Figure 25). The y-axes are scaled to the range of data plotted for each cluster, to better visualize the differences in frequency of lower abundance clusters. For this experiment, it may be interesting to additionally depict the patient information. We do this by plotting a different point shape for each patient. Indeed, we can see that often the direction of abundance changes between the two conditions are concordant among the patients.\n\n\n\nDifferent colors are used for the two conditions: unstimulated (Ref) and stimulated with BCR/FcR-XL (BCRXL). Values for each patient are indicated with different shape. The 8 cell populations are a result of manual merging of the 20 FlowSOM metaclusters.\n\nAs our goal is to compare proportions, one could take these values, transform them (e.g. logit) and use them as a dependent variable in a linear model. However, this approach does not take into account the uncertainty of proportion estimates, which is higher when ratios are calculated for samples with lower total cell counts. A distribution that naturally accounts for such uncertainty is the binomial distribution (i.e. logistic regression), which takes the cell counts as input (relative to the total for each sample). Nevertheless, as in the genomic data analysis, the pure logistic regression is not able to capture the overdispersion that is present in HDCyto data. A natural extension to model the extra variation is the generalized linear mixed model (GLMM), where the random effect is defined by the sample ID (Jia et al., 2014; Zhao et al., 2013). Additionally, in our example the patient pairing could be accounted in the model by incorporating a random intercept for each patient. Thus, we present two GLMMs. Both of them comprise a random effect defined by the sample ID to model the overdispersion in proportions. The second model includes a random effect defined by the patient ID to account for the experiment pairing.\n\nIn our model, the blocking variable is patient ID i = 1, ..., n, where n = 8. For each patient, there are ni samples measured, and j = 1, ..., ni indicates the sample ID. Here, ni = 2 for all i (one from reference and one from BCR/FcR-XL stimulated).\n\nWe assume that for a given cell population, the cell counts Yij follow a binomial distribution Yij ∼ Bin(mij, πij), where mij is a total number of cells in a sample corresponding to patient i and condition j. The generalized linear mixed model with observation-level random effects ξij is defined as follows:\n\nwhere ξij∼N(0,σξ2) and xij corresponds to the conditionBCRXL column in the design matrix and indicates whether a sample ij belongs to the reference (xij = 0) or treated condition (xij = 1). Since E(Yij \|β0, β1, ξij) = πij, the above formula can be written as follows:\n\nThe generalized linear mixed model that furthermore accounts for the patient pairing incorporates additionally a random intercept for each patient i:\n\n\n\nThe wrapper function below takes as input a data frame with cell counts (each row is a population, each column is a sample), the metadata table, and the formula, and performs the differential analysis specified with contrast K for each population separately, returning a table with non-adjusted and adjusted p-values.\n\n\n\nWe fit both of the GLMMs specified above. We can see that accounting for the patient pairing increases the sensitivity to detect differentially abundant cell populations.\n\n\n\n\n\n\n\n\n\nAn output table containing the observed cell population proportions in each sample and p-values can be assembled (and optionally written to a file).\n\n\n\n\n\nWe use a heatmap to report the differential cell populations (see Figure 26). Proportions are first scaled with the arcsine-square-root transformation (as an alternative to logit that does not return NAs when ratios are equal to zero or one). Then, Z-score normalization is applied to each population to better highlight the relative differences between compared conditions. We created two wrapper functions: normalization_wrapper performs the normalization of submitted expression expr to mean 0 and standard deviation 1, and plot_differential_heatmap_wrapper generates a heatmap of submitted expression expr_norm, where samples are grouped by condition, indicated with a color bar on top of the plot. Additionally, labels of clusters contain the adjusted p-values in parenthesis.\n\n\n\n\n\n\n\n\n\nFor this part of the analysis, we calculate the median expression of the 14 signaling markers in each cell population (merged cluster) and sample. These will be used as the response variable Yij in the linear model (LM) or linear mixed model (LMM), for which we assume that the median marker expression follows a Gaussian distribution (on the already arcsinh-transformed scale). The linear model is formulated as follows:\n\nwhere ∈ij ∼ N(0, σ2), and the mixed model includes a random intercept for each patient:\n\nwhere γi∼N(0,σγ2). In the current experiment, we have an intercept (basal level) and a single covariate, xij, which is represented as a binary (stimulated/unstimulated) variable. For more complicated designs or batch effects, additional columns of a design matrix can be used.\n\nOne drawback of summarizing the protein marker intensity with a median over cells is that all the other characteristics of the distribution, such as bimodality, skewness and variance, are ignored. On the other hand, it results in a simple, easy to interpret approach, which in many cases will be able to detect interesting changes. Another issue that arises from using a summary statistic is the level of uncertainty, which increases as the number of cells used to calculate it decreases. In the statistical modeling, this problem could be partially handled by assigning observation weights (number of cells) to each cluster and sample. However, since each cluster is tested separately, these weights do not account for the differences in size between clusters.\n\nThere might be instances of small cell populations for which no cells are observed in some samples or where the number of cells is very low. For clusters absent from a sample (e.g. due to biological variance or insufficient sampling), NAs are introduced because no median expression can be calculated; in the case of few cells, the median may be quite variable. Thus, we apply a filter to remove samples that have fewer than 5 cells. We also remove cases where marker expression is equal to zero in all the samples, as this leads to an error during model fitting.\n\n\n\nIt is helpful to plot the median expression of all the markers in each cluster for each sample colored by condition, to get a rough image of how strong the differences might be (see Figure 27). We do this by combining boxplots and jitter.\n\n\n\nDifferent colors are used for the two conditions unstimulated (Ref) and stimulated with BCR/FcR-XL (BCRXL). Values for each patient are indicated with different shape. The 8 cell populations are a result of manual merging of the 20 metaclusters.\n\nWe created a wrapper function differential_expression_wrapper that performs the differential analysis of marker expression. The user needs to specify a data frame expr_median with marker expression, where each column corresponds to a sample and each row to a cluster/marker combination. One can choose between fitting a regular linear model model = \"lm\" or a linear mixed model model = \"lmer\". The formula parameter must be adjusted adequately to the model choice. The wrapper function returns the non-adjusted and adjusted p-values for each of the specified contrasts K for each cluster/marker combination.\n\n\n\nTo present how accounting for the within patient variability with the mixed model increases sensitivity, we also fit a regular linear model. The linear mixed model has a random intercept for each patient.\n\n\n\nBy accounting for the patient effect, we detect almost twice as many cases of differential signaling compared to the regular linear model.\n\n\n\n\n\n\n\n\n\nOne can assemble together an output table with the information about median marker expression in each cluster and sample, and the obtained p-values.\n\n\n\n\n\nTo report the significant results, we use a heatmap (see Figure 28). Instead of plotting the absolute expression, we display the normalized expression, which better highlights the direction of marker changes. Additionally, we order the cluster-marker instances by their significance and group them by cell type (cluster).\n\n\n\n\n\n\n\n\n\nThe analysis of overall expression is analogous to the previous section, except that median marker expression is aggregated from all the cells in a given sample, Figure 29.\n\n\n\nDifferent colors are used for the two conditions unstimulated (Ref) and stimulated with BCR/FcR-XL (BCRXL). Values for each patient are indicated with different shape.\n\nSimilar to the analysis above, we identify more markers being differentially expressed with the LMM, which accounts for the within patient variability.\n\n\n\n\n\n\n\n\n\nAs before, we create an output table with the median marker expression calculated in each sample and the p-values, and we plot a heatmap with the significant markers sorted by their statistical significance (Figure 30).\n\n\n\n\n\n\n\n\nDiscussion\n\nIn this workflow, we have presented a pipeline for diverse differential analyses of HDCyto datasets. First, we highlight quality control steps, where aggregate characteristics of the samples are visualized (e.g. an MDS plot), allowing for verification of the experimental design, detection of batch effects and outlying samples. Next, cell population identification was carried out via clustering, which forms the basis for subsequent differential analyses of cell population abundance, differential marker expression within a population or overall marker expression differences. The approaches to differential analyses proposed here are very general and thus able to model complex experimental designs via design matrices, such as factorial experiments, paired experiments or adjustment for batch effects. We have presented a range of visualizations that help in understanding the data and reporting the results of clustering and differential analyses. The wrapper functions presented in this workflow may need to be tailored to the needs of a different experiment.\n\nClustering is one of the most challenging steps in the workflow, and its accuracy is critical to the downstream differential analyses. Getting the right resolution of clusters is crucial, since there can be situations where a biologically meaningful cell population may be differentially enriched between conditions, but in an automatic clustering, was combined with another cell population that behaves differently. While we have a good understanding of how computational algorithms recapitulate manual gating in high dimensions (Weber & Robinson, 2016), one of the open areas of research remains how to best cluster across samples. A recent approach uses a combination of high dimensional density estimation, hierarchical clustering and network inference and comparison to extract clusters across samples, with a possibility to handle batch effects (Li et al., 2017). In our approach, we aggregated all cells together before clustering. An alternative would be to cluster within each sample and then aggregate a collection of metaclusters across samples. Further research is required to better understand these effects, especially when batch effects are present.\n\nThe data analyzed here (Bodenmiller et al., 2012; Bruggner et al., 2014) was generated using sample barcoding; this strategy reduces intersample variability, since all samples are exposed to the same antibody cocktail and measured in a single acquisition (Zunder et al., 2015). Thus, the range of marker expression for each channel should, in principle, be within a similar range across samples. Additional challenges may arise when combining data from different instrument acquisitions and additional preprocessing treatments may need to be applied. Despite adjustments through bead-based normalization (Finck et al., 2013), the observed marker expression may be affected by the varying efficiency of antibody binding in each batch and by the ion detection sensitivity after machine calibration. Beyond normalization, other strategies have been proposed, such as equalizing the dynamic range between batches for each marker or the use of warping functions to eliminate non-linear distortions (see cydar vignette). However, a comprehensive evaluation of these approaches and their effect on downstream analyses is still missing. Overall, we expect that as a general rule, including batch parameters (or other covariates) in the linear modeling largely mitigates the problem.\n\nWe presented a classical statistical approach where preprocessing of the HDCyto data leads to tables of summaries (e.g. cell counts) or aggregated measurements (e.g. cluster-specific signals) for each samples, which become the input to statistical model. Of course, there are a variety of alternative computational approaches available to the user. We have mentioned citrus and CellCnn, which are both machine-learning approaches that fit a reverse model to ours (i.e. phenotype of interest as the response variable). Neither of these approaches are directly able to account for batch effects or complicated designs. However, they may have advantages in the search for rare distinguishing populations, which could be used together with our framework for formal statistical testing.\n\nWe have shown that some level of over-clustering is convenient for detecting meaningful cell populations, since automatic detection of the number of natural clusters is difficult (Weber & Robinson, 2016). However, there are tradeoffs between the resolution of clustering and the labor involved in aggregating them to biologically meaningful clusters. Overall, we take an interactive but flexible algorithm-guided approach together with subject-area experts to arrive at sensible cell populations. In particular, we rely on various visualizations, such as dendrograms, t-SNE maps or other dimension reduction techniques to guide us in the process. Alternative strategies could be combined with the statistical inference we present, such as over-clustering combined with data-driven aggregation to the optimal resolution.\n\nOne of the main goals of this workflow was to highlight how a model-based approach is able to handle complex experimental designs. This becomes important in many experimental situations where covariates (e.g. age, gender, batch) may affect the observed HDCyto data. Thus, the classical regression framework allows also to flexibly test situations well beyond two-group differences. Of course, alternatives exist for two group comparisons, such as the nonparametric Mann-Whitney-Wilcoxon test (Hartmann et al., 2016), which makes no assumptions about normality of the data, or the Student’s t-test (Pejoski et al., 2016) and its variations, such as the paired t-test.\n\nWe note that the LM, LMM and GLMM may perform poorly for extremely small samples. Solutions similar to those widely accepted in transcriptomics that share information over variance parameters (Love et al., 2014; Ritchie et al., 2015; Robinson & Smyth, 2007) could be leveraged. An example of such an approach is cydar (Lun et al., 2017), which performs the differential abundance analysis (on hypersphere counts) using the generalized linear modeling capabilities of edgeR (McCarthy et al., 2012).\n\nThe approach presented in this workflow is not fully automated due to the cluster merging, annotating, and extensive exploratory data analysis steps. In general, our philosophy is that fully automated analyses are to be avoided, but rather a battery of diagnostic checks can be designed, as we have promoted here. Cluster annotation remains a manual step in many other approaches as well. Recently, a tool was proposed for consistent characterization of cell subsets using marker enrichment modeling (MEM) (Diggins et al., 2017).\n\nTo keep the analysis of this workflow reproducible, one needs to define a random seed before running FlowSOM and t-SNE. This is especially important in the clustering step, where the order of clusters may change with different seeds, and the cluster merging needs to be matched to the seed used.\n\n\nSoftware availability\n\nAll software packages used in this workflow are publicly available from the Comprehensive R Archive Network (https://cran.r-project.org) or the Bioconductor project (http://bioconductor.org). The specific version numbers of the packages used are shown below, along with the version of the R installation. Version numbers of all Bioconductor packages correspond to release version 3.5 of the Bioconductor project. Users can install all required packages and execute the workflow by following the instructions at https://github.com/gosianow/cytofWorkflow.\n\n\n\n", "appendix": "Author contributions\n\n\n\nMN and MDR designed and ran analyses. MN drafted the manuscript with input from MDR. CK and SG performed experiments, analyzed data and gave feedback on clinical applications. CK performed the cluster merging. FJH and LMW contributed code and ideas for the analyses. MPL interpreted data and provided clinical perspective. BB gave feedback on the manuscript and the bioinformatics. All authors read and approved the final manuscript and have agreed to the content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nMN acknowledges the funding from a Swiss Institute of Bioinformatics (SIB) Fellowship.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe authors wish to thank members of the Robinson, Bodenmiller and von Mering groups from the Institute of Molecular Life Sciences, University of Zurich for helpful discussions.\n\n\nReferences\n\nAghaeepour N, Finak G, FlowCAP Consortium: Critical assessment of automated flow cytometry data analysis techniques. Nat Methods. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved, 2013; 10(3): 228–38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAngerer P, Haghverdi L, Büttner M, et al.: destiny: diffusion maps for large-scale single-cell data in R. Bioinformatics. 2016; 32(8): 1241–3. PubMed Abstract \| Publisher Full Text\n\nArvaniti E, Claassen M: Sensitive detection of rare disease-associated cell subsets via representation learning. bioRxiv. 2016. Publisher Full Text\n\nBendall SC, Davis KL, Amir el-AD, et al.: Single-cell trajectory detection uncovers progression and regulatory coordination in human B cell development. Cell. Elsevier. 2014; 157(3): 714–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBendall SC, Simonds EF, Qiu P, et al.: Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science. American Association for the Advancement of Science, 2011; 332(6030): 687–96. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBodenmiller B, Zunder ER, Finck R, et al.: Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators. Nat Biotechnol. Nature Publishing Group, 2012; 30(9): 858–67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBruggner RV, Bodenmiller B, Dill DL, et al.: Automated identification of stratifying signatures in cellular subpopulations. Proc Natl Acad Sci U S A. 2014; 111(26): E2770–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen H, Lau MC, Wong MT, et al.: Cytofkit: A Bioconductor Package for an Integrated Mass Cytometry Data Analysis Pipeline. PLoS Comput Biol. Public Library of Science, 2016; 12(9): e1005112. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDiggins KE, Greenplate AR, Leelatian N, et al.: Characterizing cell subsets using marker enrichment modeling. Nat Methods. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved, 2017; 14(3): 275–78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEllis B, Haaland P, Hahne F, et al.: FlowCore: FlowCore: Basic Structures for Flow Cytometry Data. 2017. Reference Source\n\nFinck R, Simonds EF, Jager A, et al.: Normalization of mass cytometry data with bead standards. Cytometry A. 2013; 83(5): 483–94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaghverdi L, Buettner F, Theis FJ: Diffusion maps for high-dimensional single-cell analysis of differentiation data. Bioinformatics. 2015; 31(18): 2989–98. PubMed Abstract \| Publisher Full Text\n\nHartmann FJ, Bernard-Valnet R, Quériault C, et al.: High-dimensional single-cell analysis reveals the immune signature of narcolepsy. J Exp Med. Rockefeller University Press, 2016; 213(12): 2621–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJia C, Hu Y, Liu Y, et al.: Mapping Splicing Quantitative Trait Loci in RNA-Seq. Cancer Inform. 2014; 13(Suppl 4): 35–43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKotecha N, Krutzik PO, Irish JM: Web-Based Analysis and Publication of Flow Cytometry Experiments. Curr Protoc Cytom. John Wiley & Sons, Inc. 2010; Chapter 10: Unit10.17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeipold MD: Another step on the path to mass cytometry standardization. Cytometry A. 2015; 87(5): 380–82. PubMed Abstract \| Publisher Full Text\n\nLevine JH, Simonds EF, Bendall SC, et al.: Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells that Correlate with Prognosis. Cell. Elsevier, 2015; 162(1): 184–97. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi YH, Li D, Samusik N, et al.: Scalable Multi-Sample Single-Cell Data Analysis by Partition-Assisted Clustering and Multiple Alignments of Networks. bioRxiv. Cold Spring Harbor Labs Journals. 2017. Publisher Full Text\n\nLove MI, Huber W, Anders S: Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. BioMed Central, 2014; 15(12): 550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLun AT, Richard AC, Marioni JC: Testing for differential abundance in mass cytometry data. Nat Methods. advance on (May): Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved, 2017. PubMed Abstract \| Publisher Full Text\n\nMahnke YD, Roederer M: Optimizing a multicolor immunophenotyping assay. Clin Lab Med. 2007; 27(3): 469–85, v. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcCarthy DJ, Chen Y, Smyth GK: Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res. 2012; 40(10): 4288–97. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMonti S, Tamayo P, Mesirov J, et al.: Consensus Clustering: A Resampling-Based Method for Class Discovery and Visualization of Gene Expression Microarray Data. Mach Learn. 2003; 52(1): 91–118. Publisher Full Text\n\nPejoski D, Tchitchek N, Rodriguez Pozo A, et al.: Identification of Vaccine-Altered Circulating B Cell Phenotypes Using Mass Cytometry and a Two-Step Clustering Analysis. J Immunol. American Association of Immunologists, 2016; 196(11): 4814–31. PubMed Abstract \| Publisher Full Text\n\nRitchie ME, Phipson B, Wu D, et al.: limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. Oxford University Press, 2015; 43(7): e47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobinson MD, Smyth GK: Moderated statistical tests for assessing differences in tag abundance. Bioinformatics. 2007; 23(21): 2881–7. PubMed Abstract \| Publisher Full Text\n\nRoederer M: Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry. John Wiley & Sons, Inc, 2001; 45(3): 194–205. PubMed Abstract \| Publisher Full Text\n\nSaeys Y, Gassen SV, Lambrecht BN: Computational flow cytometry: helping to make sense of high-dimensional immunology data. Nat Rev Immunol. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved, 2016; 16(7): 449–62. PubMed Abstract \| Publisher Full Text\n\nTang J, Liu J, Zhang M, et al.: Visualization Large-scale and High-dimensional Data. CoRR. abs/1602.00370; 2016. Publisher Full Text\n\nvan der Maaten L, Hinton G: Visualizing high-dimensional data using t-sne. J Mach Learn Res. 2008. Reference Source\n\nVan Gassen S, Callebaut B, Van Helden MJ, et al.: FlowSOM: Using self-organizing maps for visualization and interpretation of cytometry data. Cytometry A. 2015; 87(7): 636–45. PubMed Abstract \| Publisher Full Text\n\nvan Unen V, Li N, Molendijk I, et al.: Mass Cytometry of the Human Mucosal Immune System Identifies Tissue- and Disease-Associated Immune Subsets. Immunity. 2016; 44(5): 1227–39. PubMed Abstract \| Publisher Full Text\n\nWang B, Ramazzotti D, De Sano L, et al.: SIMLR: A Tool for Large-Scale Single-Cell Analysis by Multi-Kernel Learning. bioRxiv. Cold Spring Harbor Labs Journals. 2017. Publisher Full Text\n\nWattenberg M, Viégas F, Johnson I: How to Use t-SNE Effectively. Distill. 2016. Publisher Full Text\n\nWeber LM, Robinson MD: Comparison of clustering methods for high-dimensional single-cell flow and mass cytometry data. Cytometry A. 2016; 89(12): 1084–96. PubMed Abstract \| Publisher Full Text\n\nWilkerson MD, Hayes DN: ConsensusClusterPlus: a class discovery tool with confidence assessments and item tracking. Bioinformatics. 2010; 26(12): 1572–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao K, Lu ZX, Park JW, et al.: GLiMMPS: robust statistical model for regulatory variation of alternative splicing using RNA-seq data. Genome Biol. BioMed Central Ltd, 2013; 14(7): R74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZunder ER, Finck R, Behbehani GK, et al.: Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm. Nat Protoc. Nature Publishing Group, 2015; 10(2): 316–33. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "23055", "date": "06 Jun 2017", "name": "Aaron T. L. Lun", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nNowicka et al. describe a comprehensive workflow for a multi-sample analysis of a mass cytometry data set. They provide methods and guidelines for data processing and quality control, clustering and interpretation/visualization of the clusters. They also describe the application of statistical methods for differential analyses within clusters. The article is clear and well-written, with some opportunities for improvement that we have listed below. Overall, this will be useful resource for people looking to use R/Bioconductor for cytometry data analysis.\nMAJOR COMMENTS:\nThe principle of pooling samples prior to clustering is important to ensure that the clustering is blind to the sample labels, and thus does not bias the downstream statistical inferences. However, in data sets where the number of cells is highly variable across samples, larger samples may drive the final clustering result. The authors may consider downweighting cells from larger samples during the clustering procedure, to ensure that each sample contributes equally to the outcome.\n\nIn what scenarios is the NRS useful? In a mass cytometry experiment, the panel is explicitly designed to interrogate markers of interest, so outside of quality control it makes little sense to discard them in the analysis. Indeed, low variance contributions in PCA does not mean that the marker is not relevant, e.g., if it marks a small population.\n\nIf the expected number of cell types is not known in advance, how many metaclusters should be chosen? In very heterogeneous populations, it is easy to imagine that there may well be more than 20 distinct cell subpopulations.\n\nIn the discussion, the authors state that \"Overall, we expect that as a general rule, including batch parameters (or other covariates) in the linear modeling largely mitigates the problem.\" This is true to some extent, but will not protect the clustering from batch effects. If the batch effect shifts the intensity distribution between batches, it is possible that a subpopulation in samples of one batch is clustered with the wrong subpopulation in samples of another batch. The counts or median intensities of the cluster are inherently compromised and cannot be fixed by blocking on the batch effect in the model. In other words; when testing for changes in abundance in mass cytometry, the cells are analogous to individual reads in a transcriptomics experiment, while the vector of intensities is analogous to the genic region in which reads are counted. If the batch effect is affecting the intensities, it is analogous to changes in the definition of the genes between batches.\n\nThe authors mention using observation weights to describe the uncertainty of the median intensities when testing for differential expression of markers. We note that we have also used this approach in cydar, and it seems to work well. For differential expression of markers within each cluster, the differences in size between clusters are largely irrelevant - all else being equal, if clusters are small, the medians should be more variable, and this should be considered by the inference machinery when computing p-values.\n\nMINOR COMMENTS:\nA mention should be made of the fact that fsApply combines the intensity matrices from all data sets; this was not obvious from the code.\n\nThere are many ways to do hypothesis testing in GLMMs, with options ranging from Wald Z-tests, LRTs and parametric bootstrapping/MCMC. Some words on what glht actually does would be useful.\n\nSome of the figure captions could be explained in more detail. For example, the numbers and colouring of the entries of the heatmap in Figure 4 are presumably the median marker intensities, but this should be explicitly stated.\n\nSome minor typographical errors: \" (Angerer et al., 2016))\", \"use the flowCore [package].\"\n\nThe PBMC data set is described as \"12 different stimulation conditions\", but presumably only one was actually used (BCR/FcR-XL). This could be clarified.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "3145", "date": "14 Nov 2017", "name": "Mark Robinson", "role": "Author Response", "response": "Thank you for taking the time to read and review our paper. MAJOR POINTS: As far as we understand, downweighting of cells (e.g., from larger samples) is not currently possible with FlowSOM. We think that, in general, it may be difficult to incorporate down- weighting into existing clustering algorithms that are tailored for cytometry data, but indeed it is worth considering. One of the easy solutions to ensure that each sample contributes equally to the outcome could be down-sampling so that equal amount of cells from each sample is used in clustering. However, there are two main drawbacks of this strategy. First, a substantial amount of data (cells) may be removed from the analysis resulting in information loss. Second, during down-sampling, some of the rarer populations may become underrepresented or even skipped. Overall, it is also hard to know exactly what \"drive the clustering\" really means. We highlight these issues now in the \"Discussion\" section. NRS can be used to define new panels as it was done in Levine et al. [1]. Indeed, when there is no need for redefining the panel, it can be used as a quality control step. We mention that now in the \"Marker ranking based on the non-redundancy score\" section. In Levine et al., the NRS score was used to identify a set of surface markers that is \"sufficient\" to detect the main clusters in the AML data. As the number of markers that can be measured is limited, they could use only 16 channels for surface markers, while the remaining 15 channels were used for signaling markers. Based on average NRS, they identified the 16 surface markers, among 42, that explained the highest amount of variance in their data. The final set was slightly redefined (two markers with high scores were excluded and two markers with low scores were included) based on the biological knowledge, which agrees with the reviewer’ statement that low variance contributions in PCA do not mean that the marker is not relevant and vice versa. However, we think that NRS can still serve as a relevant guide in marker selection. The final set was then used in the panels in the following experiments. In general, as rule of thumb, we would suggest setting the number of consensus clusters (parameter maxK in ConsensusClusterPlus) to at least twice the number of expected cell populations. As this number increases, it is necessary to also increase the size of the grid in the SOM step (parameters xdim and ydim in BuildSOM). It is not necessary to know the exact number of cell types but rather the upper boundary for this number and treat that as the expected number. We also propose a strategy of re-clustering, where first main cell types are identified and extracted and then reclustered in a secondary analysis. In the updated version of our workflow, we have also added a section called \"Obtaining higher resolution\" where we describe solutions using a higher amount of clusters for higher resolution. Yes, we fully agree that including batch effects into the linear modeling does not fully protect the clustering from the batch effect. That is why it is important to account for batches already at the clustering step if possible, although this is itself still a rather open (methodological research) question. Current approaches rely on, for example, equalizing the dynamic range between batches for each marker (e.g. normalization to the 0-1 range, z-scores, quantile normalization), the use of warping functions to eliminate non-linear distortions (cydar [2]), or learning marker distribution shifts between the batches based on a manually gated reference cell type and using it to correct marker expression for the whole dataset (CellCnn [3]). A recent method called MASC [4] that, similarly to our workflow, employs mixed models for the differential abundance analysis deals with batches by identifying and excluding from the clustering analysis markers with high between-batch variability and poorly recorded cells, such as cells with extreme expression values. One could also consider, batch-wise clustering and aggregation, but these strategies also require further study. However, the effectiveness of these approaches has not been sufficiently studied, yet. We still recommend including batch information in the differential analysis as it may further help to mitigate the problem. Indeed, we agree that the size of clusters should be built into in the inference. This happens automatically in the differential abundance analysis, since we use (over-dispersed) logistic regression. In the differential marker expression analysis, where the medians are compared, one could account for the variability of medians calculated over clusters by assigning lower weights to clusters with lower cell counts. We have not done this in the current workflow, as we are still assessing the effect of it on the power and error control of the methods. MINOR POINTS: We now mention that the fsApply function, by default, combines intensity matrices from all data sets. We have now added some text explaining that the glht function uses t-tests to test the hypothesis. We have now updated the figure captions, especially those that correspond to Figures 4, 6, 7, 8, 14, 28, 30, and 32. We have fixed the identified typos. We have updated the description of the PBMC dataset. References [1] Jacob H. Levine, Erin F. Simonds, Sean C. Bendall, Kara L. Davis, El-ad D. Amir, Michelle D. Tadmor, Oren Litvin, Harris G. Fienberg, Astraea Jager, Eli R. Zunder, Rachel Finck, Amanda L. Gedman, Ina Radtke, James R. Downing, Dana Pe’er, and Garry P. Nolan. Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells that Correlate with Prognosis. Cell, 162(1):184–97, jun 2015. [2] Aaron T L Lun, Arianne C Richard, and John C Marioni. Testing for differential abundance in mass cytometry data. Nat Meth, 14(7):707–709, jul 2017. [3] Eirini Arvaniti and Manfred Claassen. Sensitive detection of rare disease-associated cell subsets via representation learning. Nature Communications, 8:14825, apr 2017. [4] Chamith Y Fonseka, Deepak A Rao, Nikola C Teslovich, Susan K Hannes, Kamil Slowikowski, Michael F Gurish, Laura T Donlin, Michael E Weinblatt, Elena M Massarotti, Jonathan S Coblyn, Simon M Helf- gott, Derrick J Todd, Vivian P Bykerk, Elizabeth W Karlson, Joerg Ermann, Yvonne C Lee, Michael B Brenner, and Soumya Raychaudhuri. Reverse Association Of Single Cells To Rheumatoid Arthritis Accounting For Mixed Effects Identifies An Expanded CD27- HLA-DR+ Effector Memory CD4+ T Cell Population. bioRxiv, 2017." } ] }, { "id": "23051", "date": "08 Jun 2017", "name": "Greg Finak", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nNowicka and colleagues present a detailed workflow for analyzing high dimensional cytometry data using open source tools within the Bioconductor framework.\n\nThe paper provides a clear path for analyzing high dimensional cytometry data, with biomarker discovery in mind, starting from raw data, through preprocessing, population discovery, annotation, and differential abundance analysis.\n\nTwo particular strengths of the proposed approach are i) the decision to use expert-guided merging of cell populations, and ii) the model-based differential abundance analysis of cell populations.\n\nThe proposed visualization and summaries of the data make i) straightforward to follow and justify, and adequate alternatives are provided and shown to perform equally well in instances where manual merging of many clusters would be cumbersome.\n\nThe modeling of cell population counts, rather than proportions, is an approach that we strongly support, and the use of logistic regression with mixed effects is a natural approach that is probably insufficiently appreciated by the community at large. That said, some of the methods proposed in the workflow have been in use in the vaccine development field for some time and should be appropriately cited. Specifically, in the section \"Visual representation with tSNE\", the authors promote coloring individual cells on a tSNE map by expression level, and later still, stratifying by condition (Fig. 11). We point the authors the article by Lin et al.1, where a very similar approach, using bioconductor tools, is undertaken to identify and visualize polyfunctional Ag-specific T-cells.\nThe discussion of existing methodological approaches to identify cytometry biomarkers associated with outcome and the discussion of modeling cell counts in favor of modeling of proportions is important, but should also reference existing work in the vaccine development field. Our group has done substantial work in this area, developing count-based models for antigen-specific T-cell response to stimulation[ref-2,3, the latter of which identified a novel biomarker of infection risk in an HIV clinical trial.\nWhile these methods do not account for covariates, they are relevant to the discussion since they utilize the Beta-binomial and Dirichlet-Multinomial distributions in a Bayesian formulation to handle over-dispersion due to subject-to-subject variability (an alternative to mixed effects modeling), and warrant mention here.\nSome additional minor points: the citation of flowCore (p5) should reference the journal publication describing the software4, since it is available, rather than the software vignette.\nFinally, note that flowCore is not used for analysis (p4), which is this context we take to mean clustering or gating, but rather is an infrastructure package that will read, write and transform cytometry data, as well as defining gate objects.\nThe core infrastructure for actually performing data-driven gating in Bioconductor is implemented in packages like flowWorkspace (Finak G, Jiang M, Gottardo R. flowWorkspace: Infrastructure for representing and interacting with the gated cytometry. 2011.) and openCyto5.\nThe citations above should be added and updated for completeness and clarity.\nOther than the above, the article is scientifically sound and the conclusions are justified by the data.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "3144", "date": "14 Nov 2017", "name": "Mark Robinson", "role": "Author Response", "response": "Thank you for taking the time to read and review our paper. Following the suggestion of the reviewer, we have incorporated the missing references, including the reference to Lin et al. [1] in the \"Visual representation with tSNE\" section, and references to MIMOSA [2] and COMPASS [3] methods in the \"Discussion\" section. We have also fixed references in the \"Data preprocessing\" section, including the reference to the flowCore package [4]. We have clarified that the packages that can be used for cell gating are flowWorkspace [5] and openCyto [6]. References [1] Lin Lin, Jacob Frelinger, Wenxin Jiang, Greg Finak, Chetan Seshadri, Pierre-Alexandre Bart, Giuseppe Pantaleo, Julie McElrath, Steve DeRosa, and Raphael Gottardo. Identification and visualization of multidimensional antigen-specific T-cell populations in polychromatic cytometry data. Cytometry Part A, 87(7):675–682, 2015. [2] Greg Finak, Andrew McDavid, Pratip Chattopadhyay, Maria Dominguez, Steve De Rosa, Mario Roederer, and Raphael Gottardo. Mixture models for single-cell assays with applications to vaccine studies. Biostatistics, 15(1):87–101, 2014. [3] Lin Lin, Greg Finak, Kevin Ushey, Chetan Seshadri, Thomas R Hawn, Nicole Frahm, Thomas J Scriba, Hassan Mahomed, Willem Hanekom, Pierre-Alexandre Bart, Giuseppe Pantaleo, Georgia D Tomaras, Supachai Rerks-Ngarm, Jaranit Kaewkungwal, Sorachai Nitayaphan, Punnee Pitisuttithum, Nelson L Michael, Jerome H Kim, Merlin L Robb, Robert J O’Connell, Nicos Karasavvas, Peter Gilbert, Stephen C De Rosa, M Juliana McElrath, and Raphael Gottardo. COMPASS identifies T-cell subsets correlated with clinical outcomes. Nat Biotech, 33(6):610–616, jun 2015. [4] Florian Hahne, Nolwenn LeMeur, Ryan R Brinkman, Byron Ellis, Perry Haaland, Deepayan Sarkar, Josef Spidlen, Errol Strain, and Robert Gentleman. flowCore: a Bioconductor package for high throughput flow cytometry. BMC Bioinformatics, 10(1):106, apr 2009. [5] Greg Finak and Mike Jiang. flowWorkspace: Infrastructure for representing and interacting with the gated cytometry, 2011. R package version 3.24.4. [6] Greg Finak, Jacob Frelinger, Wenxin Jiang, Evan W EW Newell, John Ramey, Mark MM Davis, SA Spyros a Kalams, SC Stephen C De Rosa, and Raphael Gottardo. OpenCyto: An Open Source Infrastructure for Scalable, Robust, Reproducible, and Automated, End-to-End Flow Cytometry Data Analysis. PLoS Computational Biology, 10(8):e1003806, 2014." } ] } ]	1	https://f1000research.com/articles/6-748
https://f1000research.com/articles/8-1709/v1	01 Oct 19	{ "type": "Opinion Article", "title": "Precision medicine technology hype or reality? The example of computer-guided dosing", "authors": [ "Thomas M. Polasek", "Sepehr Shakib", "Amin Rostami-Hodjegan", "Sepehr Shakib", "Amin Rostami-Hodjegan" ], "abstract": "Novel technologies labelled as ‘precision medicine’ are targeting all aspects of clinical care. Whilst some technological advances are undeniably exciting, many doctors at the frontline of healthcare view precision medicine as being out of reach for their patients. Computer-guided dosing is a precision medicine technology that predicts drug concentrations and drug responses based on individual patient characteristics. In this opinion piece, the example of computer-guided dosing is used to illustrate eight features of a precision medicine technology less likely to be hyperbole and more likely to improve patient care. Positive features in this regard include: (1) fitting the definition of ‘precision medicine’; (2) addressing a major clinical problem that negatively impacts patient care; (3) a track record of high-quality medical science published via peer-reviewed literature; (4) well-defined clinical cases for application; (5) quality evidence of benefits measured by various clinical, patient and health economic endpoints; (6) strong economic drivers; (7) user friendliness, including easy integration into clinical workflow, and (8) recognition of importance by patients and their endorsement for broader clinical use. Barriers raised by critics of the approach are given to balance the view. The value of computer-guided dosing will be decided ultimately by the extent to which it can improve cost-effective patient care.", "keywords": [ "Precision medicine", "precision dosing", "computer-guided dosing", "model-informed precision dosing", "personalized medicine", "individualized drug therapy" ], "content": "Introduction\n\nPrecision medicine is defined as ‘treatments targeted to the needs of individual patients on the basis of genetic, biomarker, phenotypic, or psychosocial characteristics that distinguish a given patient from other patients with similar clinical presentations’1. Novel technologies labelled as precision medicine are targeting all aspects of clinical care with the promise of better healthcare for all via better treatment of the individual. Examples are diverse, but include companion molecular diagnostics for pharmaco- and immuno-therapy in oncology and hematology2, pharmacogenomic-guided drug and dose selection3, and artificial intelligence to stratify clinical risk and treatment options (IBM Watson Health). Whilst some technological advances are undeniably exciting, widespread clinical application beyond specialized centers is limited, particularly outside the United States. No doubt, the words ‘precision medicine’ create business opportunities, advance academic careers, have political appeal, and resonate with the media and public. However, many doctors at the frontline of healthcare view precision medicine as merely indulgent fine-tuning for a privileged few rather than a ‘game-changer’ for all. In this opinion piece, the example of computer-guided dosing, sometimes called clinical pharmacometrics and/or model-informed precision dosing (MIPD)4,5, is used to illustrate eight features of a precision medicine technology less likely to be hyperbole and more likely to improve patient care. Barriers raised by critics of the approach are also given to balance the view.\n\nFirst, the novel technology should fit the definition of precision medicine quoted above1, rather than just applying common sense more specifically to an individual patient e.g., fitness devices linked to applications that track and encourage exercise. Computer-guided dosing predicts drug concentrations (‘exposure’) in the body based on individual patient characteristics such as age, weight and gender. Some models also incorporate physiological and molecular characteristics, including drug metabolizing enzyme and transporter activities and how these change in disease states or in the presence of interacting drugs. More sophisticated models predict drug responses based on dose-exposure-response relationships, although such modelling is relatively less advanced compared with exposure and requires further understanding of many pathophysiological states e.g., disease progression models etc. The dose required for each patient to achieve the target exposure is then relatively straightforward to determine, with the ultimate goal of accurately predicting drug responses. In cases where the pathophysiology is relatively simple or well understood (e.g., bacterial cell killing and antiviral effects), prediction of drug responses is relatively advanced6. Critics of the approach use examples where the pathophysiology is complex or poorly understood (e.g., neurology and psychiatry), which makes accurate modelling of drug responses difficult. Models can give dose predictions prior to starting drug treatment, but they are particularly powerful for dose adjustment via Bayesian feedback after initial drug exposure and/or a biomarker of response is known in a particular patient7.\n\nThe novel technology should target a well-defined clinical problem that negatively impacts patient care. Many patients receive no benefits from drug treatment. Worse still, drugs cause patient harm, costing about US $42 billion per year globally8. One of the key reasons for these problems is that drug exposure may vary more than 10-fold for the same drug at the same dose in different patients. Computer-guided dosing adjusts for between-patient variability in drug exposure. The prescribing focus changes from selecting a dose, to selecting a dose needed to achieve a target exposure, which is one-step closer to response9. This goes against the industry culture of ‘one-dose-fits-all’, which is adopted for commercial reasons. Some prescribers may also underplay the role of dose as a cause of patient harm. It is accepted that dose is just one of many factors that contribute to adverse drug effect susceptibility, including Immunological, Genetic, demographic (Age and Sex), Physiological, Exogenous factors (e.g., drug-drug interactions) and Disease and disorders (e.g., renal failure), giving the mnemonic I GASPED10. But amongst these factors, dose is the major one that can, and therefore should, be modified. Thus, the clinical problem of patient harm from drugs is more likely to be reduced, rather than ‘solved’ by computer-guided dosing.\n\nThe novel technology should not, on closer review, be so novel. Computer-guided dosing was first proposed in 1969 for anticoagulation11, with seminal publications demonstrating clinical utility in the 1970s for digoxin12. Since the millennium, affordable ‘-omics’ technologies (genomics, proteomic and metabolomics), superior analysis of biological samples, improved medical imaging, and powerful computers to analyze data, have enabled many sources of between patient variability in drug exposure and/or response to be identified, understood and then modelled e.g., warfarin. A clear narrative in the peer-reviewed literature adds confidence that a precision medicine technology is not just a ‘flash-in-the-pan’. However, a down-side of peer-reviewed literature is an over-emphasis of success stories, and the field of computer-guided dosing is probably also influenced by this publication bias.\n\nThe novel technology should have a well-defined clinical application. A classic sign of precision medicine hype is the ‘oversell’, usually from a commercial provider, which occurs when potential clinical utilities are promoted beyond the scope of actual clinical utilities. Computer-guided dosing is yet to be subject to such promotion, although several private companies have entered the market in recent years. Indeed, many doctors are unaware of computer-guided dosing or the clinical cases for which the approach is helpful. It is important to note that the patient, disease and drug characteristics that combine for high impact computer-guided dosing are well-defined4. The approach is best for difficult to dose drugs in difficult to dose patients when the clinical stakes are high. In other words, the use of narrow therapeutic index drugs when safer options are unavailable/unacceptable in pregnant women, neonates, children, those with severe organ dysfunction, the hemodynamically unstable, the frail elderly, and patients with multiple co-morbidities on polypharmacy. These patient groups are typically excluded from clinical trials that establish the recommended dose(s), so doctors are ‘flying blind’ with dosing when they must use drug treatment. For precision medicine technologies, knowing which patients, which diseases, and which drugs not to study is equally as important as knowing which clinical cases to study. This allows efficient resource allocation and the evidence of clinical utility to grow more rapidly. An important criticism of precision medicine is that it is not a ‘game-changer’ for all, however, all significant changes in clinical practice have begun in a narrow and clearly defined group of patients.\n\nThe novel technology should be supported by several independent studies that include a range of clinical, patient-reported and health economic endpoints. For computer-guided dosing, there is work on clinical outcomes with antibiotics in the critically ill, with immunosuppressants and chemotherapy in serious pediatric illnesses, and with chemotherapy in adult oncology. For example, recent randomized controlled studies comparing computer-guided dosing of paclitaxel versus body surface area-based dosing in patients with non-small cell lung cancer show trends for decreased toxicities (e.g., grade 4 hematological toxicities and neutropenia and > grade 2 neuropathy) without compromising efficacy13,14. More high quality studies of this nature are required to generate clinical evidence supportive of computer-guided dosing more broadly15. Data are especially needed for commonly used narrow therapeutic index drugs, including those started by medical specialists and continued by general practitioners, such as the direct oral anticoagulants and psychotropics (clozapine, lithium).\n\nThe novel technology should catch the eye of business developers and healthcare administrators. In the last two decades, computer-guided dosing has revolutionized drug development because it saves the pharmaceutical industry time and money7,16. The appeal of translating this success to healthcare is the economic driver for the private sector. Unsustainable public spending on healthcare is shifting the re-imbursement of drugs from a supply-based model to one that rewards positive clinical outcomes. This is forcing a re-think on the ‘one-dose-fits-all’ strategy that has previously served drug developers well. Publicly funded incentives would then be in place to find the best drug at the best dose for a particular patient. Unfortunately, strong economic drivers also create an environment for ‘sharks in the water’, further emphasizing the need for high quality evidence of benefits (section 5).\n\nThe novel technology should be embraced by doctors. Disruption to clinical workflow should be minimal and considered ‘worth the effort’ i.e., the sweet spot between clinical speed and clinical accuracy is retained. Decision support tools (DSTs) for computer-guided dosing have been integrated successfully into various in-house and commercially available electronic health records. An example with high doctor satisfaction is a DST built for busulfan, a narrow therapeutic antineoplastic drug used to prepare pediatric patients for bone marrow transplantation17. However, such examples are rare, and a major barrier to computer-guided dosing will be familiarity and acceptance of DSTs by everyday prescribers who remain unconvinced of the clinical need. A ‘forcing function’ for adoption may be the automatic inclusion of such tools within e-prescribing modules of commercially available electronic health records.\n\nFinally, the novel technology should be embraced by patients and their advocates. Broader and faster uptake of technology occurs when ‘consumers’ experience benefits and spread the word. Indeed, some precision medicine developers are now by-passing the medical profession entirely with direct-to-consumer strategies e.g., pharmacogenomic testing. There are no published data thus far on the appeal of computer-guided dosing to patients, so future studies should include endpoints to capture their perspectives5.\n\n\nConclusion\n\nMany doctors resent the insinuation of precision medicine that their clinical practice is not individualized enough and therefore not good enough. The disparity between innovative clinical applications and the reality of resource-constrained clinical practice is considered too wide. Features of precision medicine technologies more likely to bridge this disparity are illustrated here using the example of computer-guided dosing. This approach is now 50 years old and has matured to the point where clinical implementation is expected beyond specialist units in academic medical centers. The value of disruptive technologies in clinical medicine, including computer-guided dosing, will be decided ultimately by the extent to which they can improve cost-effective patient care.", "appendix": "Acknowledgements\n\nWe thank Gillian E. Caughey, BSc (Hons), PhD and Nicholas Farinola, BMedSci, BMBS, FRACP from the Department of Clinical Pharmacology, Royal Adelaide Hospital, and the University of Adelaide, for their valuable insights and comments, for which they received no compensation.\n\n\nReferences\n\nJameson JL, Longo DL: Precision medicine--personalized, problematic, and promising. N Engl J Med. 2015; 372(23): 2229–2234. PubMed Abstract \| Publisher Full Text\n\nPolasek TM, Ambler K, Scott HS, et al.: Targeted pharmacotherapy after somatic cancer mutation screening [version 2; peer review: 2 approved]. F1000Res. 2016; 5: 1551. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPolasek TM, Mina K, Suthers G: Pharmacogenomics in general practice: The time has come. Aust J Gen Pract. 2019; 48(3): 100–105. PubMed Abstract\n\nDarwich AS, Ogungbenro K, Vinks AA, et al.: Why has model-informed precision dosing not yet become common clinical reality? lessons from the past and a roadmap for the future. Clin Pharmacol Ther. 2017; 101(5): 646–656. PubMed Abstract \| Publisher Full Text\n\nPolasek TM, Shakib S, Rostami-Hodjegan A: Precision dosing in clinical medicine: present and future. Expert Rev Clin Pharmacol. 2018; 11(8): 743–746. PubMed Abstract \| Publisher Full Text\n\nKamal MA, Smith PF, Chaiyakunapruk N, et al.: Interdisciplinary pharmacometrics linking oseltamivir pharmacology, influenza epidemiology and health economics to inform antiviral use in pandemics. Br J Clin Pharmacol. 2017; 83(7): 1580–1594. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPolasek TM, Rayner CR, Peck RW, et al.: Toward Dynamic Prescribing Information: Codevelopment of Companion Model-Informed Precision Dosing Tools in Drug Development. Clin Pharmacol Drug Dev. 2019; 8(4): 418–425. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Medication Without Harm - Global Patient Safety Challenge on Medication Safety. Geneva, 2017. Reference Source\n\nPeck RW: Precision medicine is not just genomics: the right dose for every patient. Annu Rev Pharmacol Toxicol. 2018; 58: 105–122. PubMed Abstract \| Publisher Full Text\n\nFerner R, Aronson J: Susceptibility to adverse drug reactions. Br J Clin Pharmacol. 2019. PubMed Abstract \| Publisher Full Text\n\nSheiner LB: Computer-aided long-term anticoagulation therapy. Comput Biomed Res. 1969; 2(6): 507–518. PubMed Abstract \| Publisher Full Text\n\nPeck CC, Sheiner LB, Martin CM, et al.: Computer-assisted digoxin therapy. N Engl J Med. 1973; 289(9): 441–446. PubMed Abstract \| Publisher Full Text\n\nJoerger M, von Pawel J, Kraff S, et al.: Open-label, randomized study of individualized, pharmacokinetically (PK)-guided dosing of paclitaxel combined with carboplatin or cisplatin in patients with advanced non-small-cell lung cancer (NSCLC). Ann Oncol. 2016; 27(10): 1895–1902. PubMed Abstract \| Publisher Full Text\n\nZhang J, Zhou F, Qi H, et al.: Randomized study of individualized pharmacokinetically-guided dosing of paclitaxel compared with body-surface area dosing in Chinese patients with advanced non-small cell lung cancer. Br J Clin Pharmacol. 2019. PubMed Abstract \| Publisher Full Text\n\nWright DFB, Martin JH, Cremers S: Spotlight Commentary: Model-informed precision dosing must demonstrate improved patient outcomes. Br J Clin Pharmacol. 2019. PubMed Abstract \| Publisher Full Text\n\nPolasek TM, Rostami-Hodjegan A, Yim DS, et al.: What Does it Take to Make Model-Informed Precision Dosing Common Practice? Report from the 1st Asian Symposium on Precision Dosing. AAPS J. 2019; 21(2): 17. PubMed Abstract \| Publisher Full Text\n\nAbdel-Rahman SM, Breitkreutz ML, Bi C, et al.: Design and testing of an EHR-integrated, busulfan pharmacokinetic decision support tool for the point-of-care clinician. Front Pharmacol. 2016; 7: 65. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "54521", "date": "30 Oct 2019", "name": "Daniel F. B. Wright", "expertise": [ "Reviewer Expertise Model-informed precision dosing", "pharmacometrics", "clinical pharmacology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis commentary provides a well-argued and timely overview of important features of precision medicine, specifically focused on computer guided dosing. This is an important contribution at a time when model-informed precision-dosing is being touted (once again, or ‘finally’ depending on your perspective) as a means of improving patient outcomes and mitigating drug related harm. This argument is not new, but the well-considered points raised by the authors suggest that the conversation around this topic is ready to be escalated. Indeed, there is a general sense in the literature – whether real or not – that proponents of this approach are making headway with broad clinical implementation in some settings.\nIt is important to emphasize that computer-guided dosing – as used throughout the paper - is a very broad term. Outputs of clinical pharmacometric analyses or model-informed precision dosing are not synonymous with computer based dosing, as implied by the authors in the introduction, but are simply examples. Almost anything can be put on a computer as part of decision-support system, including simple empirical dosing guidelines that may have been developed historically from less than robust analyses. The authors address this point somewhat under “(1) Defined as precision medicine” but I wonder if a clear statement indicating that the paper is referring to dosing guidance that has been derived from (hopefully) robust modelling analysis is warranted. These could be simple model-based dosing tables implemented in e-prescribing systems or purpose-built Bayesian feedback programmes. As the authors note, the models underpinning the outputs might be population pharmacokinetic models or mechanistic PBPK or systems pharmacology models (there are currently few examples of the latter).\nOn a related note, the statement under “(2) addressed a clinical problem” that computer-guided dosing will adjust for between patient variability in drug exposure is not necessarily true. This is only the case of the dosing tool has been derived from a modelling analysis and/or is implemented in a Bayesian feedback system.\nThe title seems to promise some debate about whether precision medicine is hype or reality. While the paper presents eight characteristics for precision medicine that are required for “reality”, i.e. to inform clinical care, there is only a passing reference to the hype. The title might need some consideration to ensure that it aligns with the content of the paper.\nA minor point. The eight features are nicely presented but (1) and (3) could be relabelled for clarity. “Defined as precision medicine” is a bit hard to understand on first read. Something like, for example, (1) “aligns to an accepted definition of precision medicine”, (3) “Derived from high-quality medicine science” are suggestions.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "5030", "date": "17 Dec 2019", "name": "Thomas Polasek", "role": "Author Response", "response": "We thank Dr Wright for reviewing our manuscript and commenting on our work. We have now uploaded a revised copy of the manuscript with tracked changes based on the suggestions. Indeed, the revised version has benefited greatly from this input. Individual responses to each of the major comments are provided below. Comment 1 It is important to emphasize that computer-guided dosing – as used throughout the paper - is a very broad term. Outputs of clinical pharmacometric analyses or model-informed precision dosing are not synonymous with computer based dosing, as implied by the authors in the introduction, but are simply examples. ResponseWe thank Dr Wright for this suggestion and we agree. The description ‘computer-guided dosing’ was originally used instead of ‘model-informed precision dosing (MIPD)’ to appeal to a general audience less familiar with modelling and simulation in clinical pharmacology. However, after reading the review we concur that the more accurate description is superior, more focused, and less misleading. Thus, we have changed ‘computer-guided dosing’ throughout the paper to ‘model-informed precision dosing’, including in the title. Comment 2 the statement under “(2) addressed a clinical problem” that computer-guided dosing will adjust for between patient variability in drug exposure is not necessarily true. This is only the case of the dosing tool has been derived from a modelling analysis and/or is implemented in a Bayesian feedback system ResponseWe thank Dr Wright for this comment. We believe this issue has been addressed by changing ‘computer-guided dosing’ to 'model-informed precision dosing’ throughout the paper. Comment 3 The title seems to promise some debate about whether precision medicine is hype or reality. While the paper presents eight characteristics for precision medicine that are required for “reality”, i.e. to inform clinical care, there is only a passing reference to the hype. The title might need some consideration to ensure that it aligns with the content of the paper. ResponseWe thank Dr Wright for this suggestion and we agree. We have now changed the title of the manuscript to represent our position that MIPD is precision medicine technology reality not hype. Comment 4 The eight features are nicely presented but (1) and (3) could be relabelled for clarity. “Defined as precision medicine” is a bit hard to understand on first read. Something like, for example, (1) “aligns to an accepted definition of precision medicine”, (3) “Derived from high-quality medicine science” are suggestions ResponseWe thank Dr Wright for these suggestions. The titles of both of these sections have been changed accordingly." } ] }, { "id": "55228", "date": "11 Nov 2019", "name": "Richard N Upton", "expertise": [ "Reviewer Expertise Pharmacometrics", "Precision Medicine" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis seems a sensible opinion article discussing the requirements for the successful implementation of precision medicine using drug dosing as an example.\n\nTwo minor comments.\nUnder Introduction heading (1), bacterial cell killing and antiviral effects are not examples of pathophysiologies. These are examples of mechanisms of action\n\nUnder Introduction heading (6), \"Unsustainable public spending on healthcare \": Why public here? Private spending via insurance can also be unsustainable.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "5031", "date": "17 Dec 2019", "name": "Thomas Polasek", "role": "Author Response", "response": "We thank Professor Upton for reviewing our manuscript. We have now uploaded a revised copy of the manuscript with tracked changes based on the comments. Comment 1 Under Introduction heading (1), bacterial cell killing and antiviral effects are not examples of pathophysiologies. These are examples of mechanisms of action. Response We thank Professor Upton for picking up this mistake. The word ‘pathophysiology’ has been replaced with ‘mechanisms of drug action’ as suggested. Comment 2 Under Introduction heading (6), \"Unsustainable public spending on healthcare \": Why public here? Private spending via insurance can also be unsustainable. Response We thank Professor Upton for this comment. We have now added private spending to this paragraph." } ] } ]	1	https://f1000research.com/articles/8-1709
https://f1000research.com/articles/8-2111/v1	16 Dec 19	{ "type": "Brief Report", "title": "Elevated eosinophil levels observed in infantile hemangioma patients from Kaifeng, China", "authors": [ "Xianglei Li", "Chunyan Ma", "Jiaoyang Xu", "Biao Gao", "Michael Steele", "Adi Idris", "Xianglei Li", "Chunyan Ma", "Jiaoyang Xu", "Biao Gao", "Michael Steele" ], "abstract": "Infantile hemangioma (IH) is one of the most common soft-tissue neoplasms of infancy. Although clinical diagnosis for IH is well-established, the haematological parameters associated with IH are not well explored. In this short study, we observed significantly higher eosinophil (EO) numbers in IH patient blood compared to healthy controls. This contributed to the observed higher EO % in the peripheral blood of IH patients and was irrespective of age. This new haematological finding could carry a potential diagnostic/prognostic relevance for IH.", "keywords": [ "Infantile hemangioma", "eosinophil", "haematology", "China" ], "content": "Introduction\n\nInfantile hemangioma (IH) is a common benign tumour in children that presents as precursor vascular lesions, which either present at birth or develop during the early neonatal period and undergo rapid proliferation1. IH is the most common vascular tumour of infancy, occurring in up to 10% of infants2 and is characterized by high expression of genes involved in vasculogenesis, angiogenesis and tumorigenesis3. In the Chinese population, low birth weight, prematurity and maternal progesterone have been associated with IH development4. Although clinical diagnosis for IH is well-established, other than the proposed embryonic stem cell origins of IH5, little is known about the peripheral blood cell repertoire in IH patients, let alone in Chinese patients. This concise study seeks to determine any potential haematological signature(s) that may be present in the peripheral blood of IH diagnosed Chinese patients. In this retrospective study, we report significantly elevated eosinophil numbers in Chinese IH patients.\n\n\nMethods\n\nKaifeng Central Hospital (Kaifeng, China) is designated as a health care centre by the Kaifeng city government. Retrospective analysis of Kaifeng Central Hospital patient records was performed for this study and the study protocol was approved by the Kaifeng Central Hospital Ethics Committee, which waived the need for informed consent from patients/guardians for the use of their records. Underlying data are all de-identified demographic variables and blood parameters for each individual patient6. Patients’ parents/guardians had been made aware that this data could be used for research purposes.\n\nStudy subjects included paediatric patients (n = 1631) of all sexes (Male (M) = 460 / Female (F) = 1171) between the ages of 0 to 12 months (3.77 ± 2.98 months, mean ± SD) who were diagnosed with IH from January 2011 to December 2016. Control subjects (n=1602) were healthy children who had blood taken during routine medical check-up visits to the hospital during that same period. As previously seen7, we observed significantly more female IH patients than males (Chi squared test, p<0.001). The inclusion criteria included only infants up to 12 months of age and infants with all variables measured (WBC, RBC, MPV, HGB, PCT, EO%, EO#). The exclusion criteria were subjects with other existing conditions and diseases including eczema, systemic infection, allergy, haematological diseases, immunological diseases and adrenocortical insufficiency and who were not undergoing treatment for IH.\n\nPeripheral blood samples (n = 3233) were assayed for full blood panel count on the Sysmex XN-800i (Sysmex Europe GmbH, Norderstedt, Germany) as per manufacturer’s protocol. Blood variables measured included white blood cell (WBC) counts, red blood cell (RBC) counts, mean platelet volume (MPV), haemoglobin (HGB) levels, procalcitonin (PCT) levels and eosinophils (EO) percentage/counts.\n\nDue to strong non-normality of some variables the non-parametric Mann-Whitney Test was used in the analysis of continuous variables. Chi-Square test of independence was used for categorical data. All statistical analysis was done on IBM SPSS Statistics 22.0 (SPSS Institute, Chicago, IL, USA). Before analysis, all variables were reviewed for accuracy of data entry and missing values. Due to the large sample size involved, statistical analysis is focused primarily on frequencies and percentages.\n\n\nResults\n\nWe analysed blood parameters between IH patients and healthy controls (Table 1). Notably, we observed a high elevation of EO numbers in IH patients compared to healthy subjects. Compared to the healthy control (0.19±0.24 ×109/ µL), there is an almost significantly (Chi-Square test of independence, p<0.001) two-fold higher EO count in IH patients (0.4±0.37 ×109/ µL). This contributed to the observed higher EO % in the peripheral blood of IH patients.\n\nControl – Healthy subjects, IH-Infantile hemangioma patients, WBC- white blood cells, RBC- Red blood cells, MPV – Mean platelet volume, HGB- Hemoglobin, PCT-Procalcitonin, EO-Eosinophils\n\n1 Mann-Whitney Test\n\n2 Chi-Square Test of Independence\n\nThis observation was irrespective of age as significantly higher EO numbers (Mann-Whitney test, p<0.001) were observed only between IH patient and healthy control cohort for each age-matched group, not between each age group (Table 2). Other measured blood parameters were comparable between IH patients and healthy controls (Table 1).\n\n1 Mann-Whitney Test\n\n\nDiscussion\n\nElevated EOs are classically associated with the presence of inflammation in patients with conditions such as asthma, allergy and parasitic infections. Our exclusion criteria in this study discounted any possibility of this on our observations. Previous haematological analyses of blood collected from 34 IH patients in an Italian study revealed slightly elevated EO %8, but IH blood parameters were not compared to that in healthy subjects. Mean EO reference numbers in the general Chinese population are between 0.1 – 0.2 × 1099, in concordance with healthy EO levels we observe.\n\nOne major limitation in this study is the inability to compartmentalize IH patients into different clinical phases (i.e. proliferating phase, early regressing (involuting) phase, and advanced regressing (involuted) phase) as this information was not made available to us during retrospective data collection. Future work will focus on determining whether EO numbers increase progressively throughout the different IH clinical phases.\n\nPropranolol, a beta-blocking agent, has been used as the first-line therapy for the management of IH since 200810. However, propranolol use for managing IH in China only came about after findings from a prospective 2011 trial11. Given that propranolol has been shown to prevent the release of EO-activating cytokines12, propranolol would work favourably in IH patients to reduce the abnormally high EO numbers seen in our patients. In this present study, we show for the first time a significant elevation in EO numbers in IH paediatric patients and this could potentially carry a diagnostic/prognostic relevance in Chinese children. IH is commonly diagnosed clinically based on natural history of the lesion. Currently, the most important marker to accurately diagnose IH is glucose transporter 1 (GLUT1)13, though this marker is present despite the proliferative activity of the IH lesion14. The use immune cytokines as a potential biomarker for IH progression was recently proposed8,15 and some of those cytokines (e.g. interleukin -8) could directly impact EO proliferation. Standard haematological (e.g. abnormal EO numbers) and unique cytokine signatures could potentially serve as a diagnostic/prognostic marker for IH progression.\n\n\nData availability\n\nOpen Science Framework: Elevated eosinophil levels observed in infantile hemangioma patients from Kaifeng, China, https://doi.org/10.17605/OSF.IO/P8XR36.\n\nThis project contains the following underlying data:\n\n- raw data_Li et al.xls (Raw haematological data)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nLéauté-Labrèze C, Harper JI, Hoeger PH: Infantile haemangioma. Lancet. 2017; 390(10089): 85–94. PubMed Abstract \| Publisher Full Text\n\nKilcline C, Frieden IJ: Infantile hemangiomas: how common are they? A systematic review of the medical literature. Pediatr Dermatol. 2008; 25(2): 168–73. PubMed Abstract \| Publisher Full Text\n\nHarbi S, Wang R, Gregory M, et al.: Infantile Hemangioma Originates From A Dysregulated But Not Fully Transformed Multipotent Stem Cell. Sci Rep. 2016; 6(1): 35811. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen XD, Ma G, Chen H, et al.: Maternal and perinatal risk factors for infantile hemangioma: a case-control study. Pediatr Dermatol. 2013; 30(4): 457–61. PubMed Abstract \| Publisher Full Text\n\nBoscolo E, Bischoff J: Vasculogenesis in infantile hemangioma. Angiogenesis. 2009; 12(2): 197–207. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi X, Ma C, Xu J, et al.: Elevated eosinophil levels observed in infantile hemangioma patients from Kaifeng, China. OSF. 2019. http://www.doi.org/10.17605/OSF.IO/P8XR3\n\nMunden A, Butschek R, Tom WL, et al.: Prospective study of infantile haemangiomas: incidence, clinical characteristics and association with placental anomalies. Br J Dermatol. 2014; 170(4): 907–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nD'Arcangelo D, Nicodemi EM, Rossi S, et al.: Identification of serum regression signs in infantile hemangioma. PLoS One. 2014; 9(3): e88545. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu X, Zhao M, Pan B, et al.: Complete blood count reference intervals for healthy Han Chinese adults. PLoS One. 2015; 10(3): e0119669. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLéauté-Labrèze C, Dumas de la Roque E, Hubiche T, et al.: Propranolol for severe hemangiomas of infancy. N Engl J Med. 2008; 358(24): 2649–51. PubMed Abstract \| Publisher Full Text\n\nJin YB, Lin XX, Ye XX, et al.: [A prospective study of propranolol as first-line treatment for problematic infantile hemangioma in China]. Zhonghua Zheng Xing Wai Ke Za Zhi. 2011; 27(3): 170–3. PubMed Abstract\n\nHallsworth MP, Twort CH, Lee TH, et al.: beta(2)-adrenoceptor agonists inhibit release of eosinophil-activating cytokines from human airway smooth muscle cells. Br J Pharmacol. 2001; 132(3): 729–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNorth PE, Waner M, Mizeracki A, et al.: GLUT1: a newly discovered immunohistochemical marker for juvenile hemangiomas. Hum Pathol. 2000; 31(1): 11–22. PubMed Abstract \| Publisher Full Text\n\nNorth PE, Waner M, James CA, et al.: Congenital nonprogressive hemangioma: a distinct clinicopathologic entity unlike infantile hemangioma. Arch Dermatol. 2001; 137(12): 1607–20. PubMed Abstract \| Publisher Full Text\n\nYamashita T, Jinnin M, Makino K, et al.: Serum cytokine profiles are altered in patients with progressive infantile hemangioma. Biosci Trends. 2018; 12(4): 438–41. PubMed Abstract \| Publisher Full Text" }	[ { "id": "57886", "date": "02 Jan 2020", "name": "Mark I.R. Petalcorin", "expertise": [ "Reviewer Expertise clinical chemistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this concise article, Li and co-authors reported that the infantile hemangioma (IH) patients showed significantly higher eosinophil levels more than the healthy control subjects suggesting a potential diagnostic relevance. Although promising, the article requires more in-depth analysis especially in addressing the causation factors that result in the increase of eosinophil levels.\nI have some concerns on the data analysis. Firstly, the mean values of percent eosinophils observed in IH patients of 3.96 and in healthy subjects of 1.91, as shown in Table 1, are both still within the normal clinical range of 0-6% EO (Medscape). Thus, the elevated eosinophil levels in IH patients might be interpreted as physiologically irrelevant by clinicians as the values are still within the normal range.\nSecondly, whilst the difference of %EO values between IH and healthy subjects is statistically significant, the raw data show that only 15% of the total IH patients and about 5% of the healthy subjects have high %EO values above the normal range of 6%. It would be useful if the authors will include this in their analysis considering the clinical implications. In addition, the authors should also specify what is the normal range of %EO used in China as this can vary in different clinical laboratories.\nLastly, I think that there are other factors contributing to the observed increase of eosinophil levels that might be present but not measured in this study such as drug treatment given to IH patients, which could be the underlying cause of the increase but not taken into account. This is the limitation of a retrospective study such as this, in which the authors have no control on how the data were designed and collected, and whether the patients received drug treatment or not. The claim of a potential diagnostic usefulness for this study is an overestimation as the causation is not well-established but only through the association. But this is a good pilot study to test the hypothesis that can be further explored.\nAs a minor comment, it would be useful to include the units used for all the parameters mentioned in the raw haematological data.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "57885", "date": "27 Jan 2020", "name": "Swaminathan Sethu", "expertise": [ "Reviewer Expertise Immunology", "Immunophenotyping", "Immune cell subset variations in health and disease" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe following are minor suggestions that may improve the clarity and interpretation of the data\nThe title can be “Elevated peripheral blood eosinophil levels in infantile hemangioma patients” or “Elevated peripheral blood eosinophil levels in Chinese patients with infantile hemangioma”.\n\nThe authors have pointed out that IH is significantly higher in females compared to males. It would be useful to analyse and represent the results in table 1 and 2 based on gender. In other words, in addition to the current statistical analysis, it would be interesting to know whether the eosinophil (EO) levels were significantly different between controls and IH in males and females subjects separately. The authors can also expand the Table 1 parameters based on gender as well.\n\nFurther, it will be useful to know the normal range for EO in pediatric population. The authors have mentioned the range for Chinese adult population. The authors can calculate the proportion (%) of subjects with IH above the normal range (if available) or the proportion (%) of subjects with IH above the median level in the control group. The authors can also attempt AUC analysis, if possible to improve the clinical relevance of EO levels in the diagnosis of IH.\n\nIt would be useful to know whether the authors had access to the proportions of other leukocyte subsets other than EO. This would be relevant and the authors can consider addressing this in the discussion.\n\nIt is unclear how the % of Procalcitonin (PCT) was computed and whether it is possible to include the concentration range for the same. Further, it was not stated as to why PCT was included in the inclusion criteria and how it is relevant to IH or EO levels in the context of IH.\n\nCould power calculation be done to show the robustness of the finding with relevance to the sample size?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-2111
https://f1000research.com/articles/8-2110/v1	16 Dec 19	{ "type": "Case Report", "title": "Case Report: Malignant Thymoma And Seronegative Myasthenia Gravis", "authors": [ "Cláudia Sousa", "Mafalda Cruz", "Ana Neto", "Kayla Pereira", "Mónica Henriques", "Rui Marques", "Paula Alves", "Mafalda Cruz", "Ana Neto", "Kayla Pereira", "Mónica Henriques", "Rui Marques", "Paula Alves" ], "abstract": "Myasthenia gravis (MG) is present in 50% of thymomas and is rarely associated with thymic carcinoma. We present the case of a 49-year-old woman with malignant thymoma, treated with surgery followed by radiotherapy, and a late seronegative MG diagnosis. This case reports the importance of a multidisciplinary approach to the management of the potential correlation of malignant and benign diseases.", "keywords": [ "Seronegative myasthenia gravis", "malignant thymoma", "tomotherapy", "paraneoplastic disorder." ], "content": "Introduction\n\nMalignant thymoma is a rare epithelial neoplasm and accounts for 30% of anterior mediastinal tumors. The highest incidence is in the 7th decade of life and is more common in men. About one-third of patients are asymptomatic at diagnosis, which is commonly found as incidentaloma. Of the symptomatic patients, 60% presents with a parathymic syndrome manifestation and 40% has symptoms relating to impingement by intrathoracic mass1–3. Myasthenia Gravis (MG) has the highest incidence among the parathymic syndromes and occurs in 50% of patients with thymoma. Of patients with MG, 15% have a thymoma and 60% will have thymic lymphoid hyperplasia1,4. As an autoimmune disorder, 85% of these patients have autoantibodies directed against postsynaptic nicotinic acetylcholine receptor (AChR). Thymectomy is the main treatment modality and complete resection is an important prognostic factor, with locoregional relapse reduction and possible resolution of MG symptoms. If high risk factors are present, radiotherapy should be considered as adjuvant treatment1.\n\n\nCase presentation\n\nA 47-year-old caucasian woman, businesswoman, without important medical history, presented with fatigue and occasional dyspnea in May 2016. A computed tomography (CT) scan was performed and showed a pre-vascular mass in the anterior mediastinum with 7 centimeters of axial axis with features of a malignant thymoma. A 18F-fluorodeoxyglucose positron emission tomography integrated with computer tomography (18F-FDG PET/CT) showed a hypermetabolic mediastinal mass, suggestive of high-grade neoplasia. The remaining diagnostic investigation was normal, without alterations of autoantibodies directed against postsynaptic nicotinic acetylcholine receptor (AChR), thyroid hormones, β-human chorionic gonadotropin (β-HCG) and α-fetoprotein. The patient underwent incomplete surgical resection due to tumor adherence to the brachiocephalic venous trunk. The histological study revealed a WHO type B1 in IIA stage of Modified Masaoka. The patient was referred for adjuvant radiotherapy and received 54Gy (1.8Gy/fraction) over the tumor bed and a total of 60 Gy (2Gy/fraction) in 30 fractions over residual tumor, by tomotherapy (Figure 1). During treatment the patient maintained an excellent general status, without relevant toxicity. One year after radiotherapy, the patient revealed severe worsening of fatigue with muscle weakness in the upper limbs, dysphagia and right diplopia. Since AChR antibodies were negative, fibromyalgia was considered. Due to maintenance of suspected MG, she started pyridostigmine 180 mg per day and presented fatigue improvement, but poor tolerance. Currently in the 33rd month of clinical control, the patient is medicated with deflazacort 6 mg, fluoxetine 20 mg, alprazolam 0.5 mg, trazodone 100 mg and maintains a stable symptomatic condition with no signs of disease recurrence (Figure 2). Surveillance continues through chest CT every 6 months and regular evaluation in radiation oncology, pneumology, psychiatry, neurology and neuro-ophthalmology.\n\n\nDiscussion\n\nParaneoplastic neurological degenerations (PND’s) affects 1 in 10,000 patients with cancer4. MG is included and is often associated with pathological abnormalities of the thymus. Given the incidence, when a diagnosis of thymoma is suspected, further investigation should be undertaken to exclude MG1. A problem of seronegative MG has been the lack of a gold standard in its diagnosis and, it is known that, the proportion of these patients varies from 5% to 30% among studies5. Seronegative MG has been reported in a few cases of benign thymoma and in only one case of malignant thymoma6. It should be noted that autoantibodies against muscle-specific kinase (MuSK) are present in about 10% of the all cases of MG and in 30% of AChR seronegative MG7. In our case, the diagnosis of MG was supported on clinical and pharmacological basis. We should be aware that, besides autoantibodies against AChR, other antibodies can be associated with this disease, including autoantibodies against MuSK and lipoprotein-related protein 4 (LRP4), however these were not tested. Additionally, new assays could improve the sensitivity in antibody detection7.\n\nGenerally, the highest incidence of malignant thymoma is in the 7th decade of life, but for patients with MG, the peak of incidence is in the 4th decade, as observed in this case1.\n\nThe differential diagnosis allowed the exclusion of neuroendocrine, germinative, hematologic and pulmonary tumors, which is crucial for the treatment plan. Surgery is the main treatment for malignant thymoma and 85% of stage II tumors are resectable. Complete resection is an important prognostic factor. In these patients, thymectomy can also be considered as an effective treatment for MG with symptomatic improvement in 50% of cases. This response seems to be associated with high AChR activity1. The efficacy of thymectomy in the treatment of MG is noted in the MGTX trial, showing that local control allows less dependence on immunosuppressive medication and less exacerbations requiring hospitalization8.\n\nAdjuvant RT is associated with better disease free-survival (DFS) with an impact on the overall survival of stage II and III tumors with a positive margin1. In this case, with incomplete resection, RT is considered to be mandatory for satisfactory local control. Advanced RT techniques, such as tomotherapy, that allows the combination of intensity-modulated radiotherapy (IMRT) and image-guided radiotherapy (IGRT), with helical radiation deposition, can provide high tumor control rates and a satisfactory toxicity profile1.\n\nIn our case, the patient showed worsening of neurological symptoms after the thymectomy and adjuvant RT. At that moment, fibromyalgia was considered in the absence of autoantibodies against AChR. Characterized by widespread musculoskeletal pain, fatigue and sleep disorder, fibromyalgia is more frequent in women, especially if other autoimmune disorders are present. MG is a differential diagnosis of fibromyalgia, as it is associated with post-exercise and generalized fatigue but not coupled with widespread pain, a symptom that our patient did not present9,10.\n\nSome studies report the importance of assuring clinical stability with immunosuppression prior to surgery in order to prevent rapid perioperative deterioration7. Given the late diagnosis, our patient only started corticotherapy after thymectomy and the systemic progression of the immunological disease.\n\nIt is possible that, in this case, the early diagnosis with initiation of immunosuppressive therapy, especially before surgery, could have changed the course of the disease1,8.\n\nIn conclusion, given the impact of an earlier diagnosis of MG on quality of life, when thymoma is suspected, seronegativity should prompt further investigated rather than result in MG exclusion.\n\n\nPatient consent\n\nObtained.\n\n\nLicense\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Author contributions\n\n\n\nDrafting of the manuscript: CS. Critical revision of the manuscript for important intellectual content: CS, MC, AN, KP, RM, MH and PA.\n\n\nReferences\n\nPerez C, et al.: Principles and Practice of Radiation Oncology. 2018.\n\nWilkins kB, Sheikh E, Green R, et al.: Clinical and pathologic predictors of survival in patients with thymoma. Ann Surg. 1999; 230(4): 562–72; discussion 572–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRomi F: Thymoma in myasthenia gravis: from diagnosis to treatment. Autoimmune Dis. 2011; 2011: 474512. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlbert ML, Darnell RB: Paraneoplastic neurological degenerations: keys to tumour immunity. Nat Rev Cancer. 2004; 4(1): 36–44. PubMed Abstract \| Publisher Full Text\n\nVincent A, Bowen J, Newsom-Davis J, et al.: Seronegative generalised myasthenia gravis: clinical features, antibodies, and their targets. Lancet Neurol. 2003; 2(2): 99–106. PubMed Abstract \| Publisher Full Text\n\nRichards J, Howard JF Jr: Seronegative myasthenia gravis associated with malignant thymoma. Neuromuscul Disord. 2017; 27(5): 417–418. PubMed Abstract \| Publisher Full Text\n\nLee JI, Jander S: Myasthenia gravis: recent advances in immunopathology and therapy. Expert Rev Neurother. 2017; 17(3): 287–299. PubMed Abstract \| Publisher Full Text\n\nWolfe GI, Kaminski HJ, Sonnett JR, et al.: Randomized Trial of Thymectomy in Myasthenia Gravis. 2016; 8(12): E1782–E1783. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOkifuji A, Gao J, Bokat C, et al.: Management of fibromyalgia syndrome in 2016. 2016; 6(4): 383–400. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaltsas G, et al.: Fibromyalgia.Endotext [Internet]. South Dartmouth (MA): MDText.com [Internet]. 2017. Reference Source" }	[ { "id": "57996", "date": "27 Dec 2019", "name": "Bruno Fionda", "expertise": [ "Reviewer Expertise Radiation Oncology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors presented a very interesting care report about malignant thymoma and seronegative myasthenia gravis. The case is presented with sufficient clinical details and the discussion included highlights the importance of the findings and their relevance to future understanding of disease. However, in my opinion, there some points which might be empowered especially regarding the details provided about physical examination and diagnostic tests. In particular the conclusion of the abstract where the authors state \"This case reports the importance of a multidisciplinary approach to the management of the potential correlation of malignant and benign diseases\" is not sufficiently supported in the case presentation. I would suggest to implement the description of the multidisciplinary management including the role of the other specialists (eg. neurologists) that the authors believe to be useful. I would stress the concept of multidisciplinarity also in the discussion (and not only in the abstract).\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "94313", "date": "13 Oct 2021", "name": "Nguyen Minh Duc", "expertise": [ "Reviewer Expertise Radiology and Oncology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIs the background of the case’s history and progression described in sufficient detail? The case’s history and progression had enough information.\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Please revise Figure 1 by cutting the outline which contained redundant information. Only introducing figure of disease like Figure 2. Please also add the asterisk or arrow in the Figures 1 and 2 along with legend to clearly describe the lesion and information related to them.\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Enough information.\nIs the case presented with sufficient detail to be useful for other practitioners? Yes.\nThis manuscript is about a case of malignant thymoma accompanied by myasthenia gravis. Please compare and discuss with the two below up-to-date references to make your manuscript better and more informative.\n1) Tuan PA, Duc NM: Magnetic resonance imaging characteristics of thymoma in Vietnamese patients with myasthenia gravis in relation to histopathological type and disease staging.Contemp Oncol (Pozn). 2020; 24 (3): 193-199 2) Tuan PA, Minh Duc N: Ectopic thymoma in the middle mediastinum: A case report and literature review.Clin Ter. 2021; 172 (2): 94-98\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "94308", "date": "18 Oct 2021", "name": "Y Muralidhar Reddy", "expertise": [ "Reviewer Expertise Neuromuscular disorders", "Neuroimmunology", "Neurosonology", "Stroke and cerebrovascular diseases", "Neuroinfections" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors described an interesting case of a middle-aged woman who developed seronegative myasthenia gravis following the partial resection and radiotherapy of malignant thymoma. These are my comments about this paper.\n\nDiagnosis of myasthenia gravis in the patient described in this report was based on the resolution of symptoms with pyridostigmine. One or more of the following tests are necessary to confirm this: 1) decrement response on repetitive nerve stimulation 2) jitter or block in craniofacial or upper limb muscles on single-fibre electromyography 3) resolution of diplopia on ice-pack test 4) resolution of symptoms after administration of intravenous edrophonium or neostigmine.\n\nI suggest including the findings of these tests in the case description and mention the reasons if these tests were not performed in the revised version.\n\nApart from Anti-acetylcholine receptor antibodies, thymomatous myasthenia gravis is also associated with many other antibodies directed against the postsynaptic membrane proteins such as Muscle-specific kinase (MuSK), Low-density lipoprotein receptor protein 4 (LRP-4), Ryanodine receptor and Titin. Moreover, the positivity rate varies with the technique of antibody assay. Cell-based assays are most sensitive. Therefore, I would suggest mentioning the assay technique used to test anti-acetylcholine receptor antibodies and the results of other antibody testing if at all performed. I would also suggest adding a note in the discussion section regarding false-negative results with various assay techniques.\nAuthors could refer to these articles for these aspects and quote them also in the revised version:\nPark KH, Waters P, Woodhall M, Lang B, Smith T, Sung JJ, Kim KK, Lim YM, Kim JE, Kim BJ, Park JS, Lim JG, Kim DS, Kwon O, Sohn EH, Bae JS, Yoon BN, Kim NH, Ahn SW, Oh J, Park HJ, Shin KJ, Hong YH. Myasthenia gravis seronegative for acetylcholine receptor antibodies in South Korea: Autoantibody profiles and clinical features. PLoS One. 2018 Mar 8;13(3):e0193723. doi: 10.1371/journal.pone.0193723.\nLazaridis K, Tzartos SJ. Autoantibody Specificities in Myasthenia Gravis; Implications for Improved Diagnostics and Therapeutics. Front Immunol. 2020 Feb 14;11:212. doi: 10.3389/fimmu.2020.00212.\n\nFibromyalgia cannot be considered as a differential diagnosis for the symptom complex which the patient developed following thymectomy. The absence of pain accompanied by double vision and dysphagia negate fibromyalgia. On the other hand, polyneuritis cranialis and radiation brachial plexitis could be more appropriate to consider in the given clinical setting. MR contrast of brain and nerve conduction studies are done to confirm these. I would suggest the authors discuss these diseases in the differential diagnosis section of the case presentation and how they were ruled out supported by relevant clinical findings and investigations.\n\nIn clinical research, myasthenia gravis is classified as per the myasthenia gravis foundation of America (MGFA), and therapeutic response is classified as per MGFA post-intervention status (PIS). I suggest that the authors describe the patient status at diagnosis and after therapy using this terminology in the revised version.\nAuthors may refer to the following articles for these aspects:\nJaretzki A 3rd, Barohn RJ, Ernstoff RM, Kaminski HJ, Keesey JC, Penn AS, Sanders DB. Myasthenia gravis: recommendations for clinical research standards. Task Force of the Medical Scientific Advisory Board of the Myasthenia Gravis Foundation of America. Neurology. 2000 Jul 12;55(1):16-23. doi: 10.1212/wnl.55.1.16.\n\nThe surgical details are not mentioned in the case description. Was it an open surgery or video-assisted thoracoscopy surgery? Although limited resection of thymoma was done due to vascular invasion, in this case, the surgical technique and completeness of the surgery have clinical relevance in any patient who develops myasthenia gravis following thymectomy.\nI would suggest authors describe, therefore, the relevant surgical aspects.\n\nI would suggest discussing in more detail the clinical course, antibody profiles and outcomes of seronegative and double seronegative myasthenia gravis in the discussion section.\nAuthors may refer to and quote the following articles:\nCortés-Vicente E, Gallardo E, Martínez MÁ, Díaz-Manera J, Querol L, Rojas-García R, Illa I. Clinical Characteristics of Patients With Double-Seronegative Myasthenia Gravis and Antibodies to Cortactin. JAMA Neurol. 2016 Sep 1;73(9):1099-104. doi: 10.1001/jamaneurol.2016.2032.\nVincent A, Bowen J, Newsom-Davis J, McConville J. Seronegative generalised myasthenia gravis: clinical features, antibodies, and targets. Lancet Neurol. 2003 Feb;2(2):99-106. doi: 10.1016/s1474-4422(03)00306\nJonathan Morena, Samantha LoRusso, Bakri Elsheikh, Miriam Freimer, Chad Hoyle, Benjamin Jiang, William Arnold. Seronegative Myasthenia Gravis: A Retrospective Review of the Clinical Characteristics at a Large Academic Center (2769). Neurology Apr 2021, 96 (15 Supplement) 2769.\n\nWhy was the patient not started on steroid-sparing immunotherapy? Why the patient still using trazodone, fluoxetine and alprazolam even after fibromyalgia was ruled out? It would suggest that the authors add a comment explaining these therapeutic decisions.\n\nHighlight of this case is the worsened myasthenia after resection of thymoma. Therefore I suggest discussing risk factors and the course of post-thymectomy myasthenia in the discussion section.\nAuthors can refer to and quote the following articles for this purpose:\nMyasthenia Gravis Appearing After the Removal of Thymoma Lewis P. Rowland, Henry Aranow, Paul F. A. Hoefer Neurology Aug 1957, 7 (8) 584; DOI: 10.1212/WNL.7.8.584 Kim A, Choi SJ, Kang CH, Lee S, Son H, Kim JA, Shin JY, Kim SM, Hong YH, Sung JJ. Risk factors for developing post-thymectomy myasthenia gravis in patients with thymoma. Muscle Nerve. 2021 Apr;63(4):531-537. doi: 10.1002/mus.27169.\nMineo TC, Tamburrini A, Schillaci O, Ambrogi V. Onset and Evolution of Clinically Apparent Myasthenia Gravis After Resection of Non-myasthenic Thymomas. Semin Thorac Cardiovasc Surg. 2018 Summer;30(2):222-227. doi: 10.1053/j.semtcvs.2018.02.027.\nYamada Y, Yoshida S, Iwata T, Suzuki H, Tagawa T, Mizobuchi T, Kawaguchi N, Yoshino I. Risk factors for developing postthymectomy myasthenia gravis in thymoma patients. Ann Thorac Surg. 2015 Mar;99(3):1013-9. doi: 10.1016/j.athoracsur.2014.10.068.\nSun XG, Wang YL, Liu YH, Zhang N, Yin XL, Zhang WJ. Myasthenia gravis appearing after thymectomy. J Clin Neurosci. 2011 Jan;18(1):57-60. doi: 10.1016/j.jocn.2010.05.018.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-2110
https://f1000research.com/articles/8-2109/v1	16 Dec 19	{ "type": "Review", "title": "Pharmacovigilance in perspective: drug withdrawals, data mining and policy implications", "authors": [ "Muaed Alomar", "Subish Palaian", "Moawia M. Al-tabakha", "Subish Palaian", "Moawia M. Al-tabakha" ], "abstract": "Considering that marketed drugs are not free from side effects, many countries have initiated pharmacovigilance programs. These initiatives have provided countries with methods of detection and prevention of adverse drug reactions at an earlier stage, thus preventing harm occurring in the larger population. In this review, examples of drug withdrawals due to effective pharmacovigilance programs have been provided with details. In addition, information concerning data mining in pharmacovigilance, an effective method to assess pharmacoepidemiologic data and detecting signals for rare and uncommon side effects, is also examined, which is a method synchronized with information technology and advanced electronic tools. The importance of policy framework in relation to pharmacovigilance is discussed in detail, and country experiences upon implementation of pharmacovigilance policies is highlighted.", "keywords": [ "Pharmacovigilance", "data mining", "drug withdrawals", "pharmacovigilance policies" ], "content": "Introduction\n\nPharmacovigilance (PV) has been a valuable method in identifying adverse drug reactions (ADRs) and improving the safe use of medicines1. PV has been the backbone for many drug safety interventions, such as drug withdrawals, labelling changes and prescription restrictions2–4. The advancement and synchronization of information technology had led to valuable contributions in signal detection and data mining processes in PV5. It is important to have policy framing to incorporate PV measures in every country’s drug regulatory mechanisms, so as to implement and sustain drug safety monitoring processes. In this review article, three important aspects of PV are discussed; significant examples of drug withdrawals as an outcome of PV data, data mining and its role in PV, and policy implications related to PV. In addition, PV experiences in selected countries are detailed.\n\n\nExamples of drug withdrawals as a result of pharmacovigilance\n\nMany medications have been withdrawn from the market due to their severe, harmful or life-threatening effects. Following marketing approval, once the first ADRs are reported, the reports will be analyzed and the incident will be investigated; and if post marketing surveillance indicates harmful effects for the medication, it will be withdrawn from the market6.\n\nRofecoxib (Vioxx), manufactured by Merck & Co. in 1999, was indicated as an NSAID in the treatment of “osteoarthritis, rheumatoid arthritis, acute pain and menstrual pain”. In the marketing stages, the company did not mention any cardiovascular disorders. Between 2000 and 2002, reports started to emerge on the hazards of Vioxx, but it took a 3-year clinical trial, “APPROVe” (Adenomatous Polyp Prevention of Vioxx), executed by Merck Frosst Canada, which lead to participants experiencing cardiovascular events such as heart attacks and strokes, for the drug to be withdrawn from the market7. In 2004, during a US Senate hearing concerning rofecoxib issues, Dr. David Graham, who was the associate director in the U.S. Department of Food and Drug Administration (FDA)’s Office of Drug Safety, stated: “The approval of rofecoxib (Vioxx) by the US FDA has led to the single greatest drug safety catastrophe in the history of this country or the history of the world”. It has been estimated that 88,000 to 139,000 Americans have had heart attacks or strokes due to Vioxx. The drug was finally discontinued in 20048.\n\nLysergic acid diethylamide (LSD), was discovered in 1938 by Dr. Hofmann, who was in Sandoz Laboratories in Switzerland9. After five years, it became evident that the drug was causing hallucinations, euphoria, delusions, depression, as well as suicidal thoughts10,11. In 1970, LSD was placed in Schedule 1 category of drugs; the most restricted category, following passage of the Controlled Substances Act. This category indicates “a high potential for abuse, no recognized medical use, and no safety when used by a physician”9.\n\nBenfluorex (Mediator) was first manufactured and marketed by Servier in 1976 in France as an add-on therapy for hyperlipidemia and diabetes associated with obesity. In 1998, an official PV investigation was opened regarding the drug in France due to its “potential danger”, and Italian regulators expressed apprehension to the European Medicines Agency (EMA). In 1999, two cardiovascular complications were reported in France. In 2003, Spanish regulators reported a cardiac valvulopathy case to the EMA12. Servier decided to withdraw the drug from Spain and Italy through not renewing their expired license13. In 2009, AFSSAPS (Agence Française de Sécurité Sanitaire des Produits de Santé), the regulatory authority of France14, suspended benfluorex marketing due to “efficacy and safety issues” especially after a case control study that was done by a chest physician, Irene Frachon, in which she discovered that drug-induced valvular heart disease is associated with benfluorex. Servier withdrew the medication worldwide after earning 20 million Euros each year for the previous 15 years12. EMA fully withdrew the drug in 201013.\n\nSibutramine (Meridia, US; Reductil, UK)8, a weight management and weigh loss agent, was approved in Europe in 199915, and in other parts of the world. Since 2002, many cardiovascular events were reported, including hypertension, tachycardia, arrhythmia and myocardial infarction (MI). In Sibutramine Cardiovascular Outcomes Trial (SCOUT), results demonstrated that patients with preexisting cardiovascular disease who had taken sibutramine had an increased likelihood of developing MI or stroke16. David Graham - in his testimony before the Senate committee about medications that may harm the patients - included Meridia as one of them8. Sibutramine was withdrawn from the US and European markets in 201015,16.\n\nPergolide (Permax) was developed by Eli Lilly & Co., and approved in 1988 for the management of Parkinson’s disease symptoms (Reuters, 2007)17. It was then withdrawn from the US market in 2007 due to “increased rates of cardiac valvular dysfunction (cardiac valvulopathy)”6.\n\nPemoline (Cylert) was approved in Europe in the sixties, and in the US in 1975, for the treatment of attention deficit hyperactivity disorder (ADHD). Between 1975 and 1989, the FDA received reports of “12 cases of jaundice and 6 deaths in youths ascribed to pemoline hepatotoxicity”. Serious hepatotoxicity was unveiled only in 1996. Pemoline hepatotoxicity reporting was not sufficient in the 1980s to stir an action, due to the poor post marketing surveillance and reporting systems18. Following those incidences, the FDA mandated a black box warning to highlight the risk of hepatotoxicity. Afterwards, another case of liver failure was reported, forcing the manufacturer of Cylert (Abbott Laboratories) to cease production in May 2005, and the FDA notified healthcare professionals about the discontinuation of all pemoline products19.\n\nThe Contergan scandal took place in early 1960s. Contergan had thalidomide as the active ingredient, a product of a German company “Chemie-Grünenthal”, having sedative effects, apparently non-toxic, with few side effects20. It was used as a sedative, hypnotic and antiemetic for pregnant women21. Mothers who used thalidomide in their early pregnancy had horrific results, as over 10,000 children were born with birth defects such as phocomelia22. It was withdrawn from most markets between 1961 and 196221.\n\nValdecoxib (Bextra) was an NSAID used for arthritis and joint pain23. According to The New England Journal of Medicine, increased cardiovascular events occurred after coronary artery bypass grafting surgery associated with valdecoxib usage24. Cardiovascular complications were reported, as well as “Stevens–Johnson syndrome, erythema multiforme, and toxic epidermal necrolysis”. These reports led to valdecoxib’s withdrawal from the market in 200523.\n\nLevamisole (Ergamisol) is an immunomodulatory agent used as an adjunct chemotherapeutic drug. It was manufactured by Janssen Pharmaceutica in 1966. Serious side effects were discovered, including: “neutropenia, agranulocytosis, cutaneous vasculopathy, and leukoencephalopathy”25. Levamisole was withdrawn from the US market in 2000. However, it is currently being used as an antihelminthic in veterinary medicine and can be found in street drugs as a cocaine adulterant26.\n\nHydromorphone hydrochloride extended release (Palladone) manufactured by Purdue Pharma and launched in the USA in January 2005, was used as a narcotic analgesic27. The FDA withdrew the drug in July of the same year due to dose dumping with alcohol, which leads to accidental overdosing27,28. Dose dumping is the release of large amount of hydromorphone drug from the extended release form leading to toxicity28.\n\nCisapride (Propulsid), manufactured by Janssen Pharmaceutica29, was indicated as a prokinetic for severe heartburn associated with gastroesophageal reflux disease. Post marketing studies showed patients experiencing “palpitations, unusual tachyarrhythmia, torsades de pointes, ventricular fibrillation, QT prolongation, and sudden death”30. Cisapride was associated with 341 heart rhythm abnormalities cases and 80 deaths. Most of these cases involved patients taking other medications or have medical conditions that increased cardiac arrhythmia risk29. It was later withdrawn in 200030.\n\nDrotrecogin alfa (Xigris) is a recombinant human activated protein C that has anti-thrombotic, profibrinolytic and anti-inflammatory activity31. It was indicated for treatment of severe sepsis. Xigris was marketed by Eli Lilly; the FDA approved it in 200131,32, while the EMA approved it in 200232. Following a study (PROWESS-SHOCK) that included 1696 patients and concluded that Drotrecogin alfa did not reduce mortality within 28 days, Eli Lilly voluntarily withdrew Xigris from the market in October 2011, and the FDA and EMA communicated the decision to healthcare professionals33,34.\n\nAprotinin (Trasylol), manufactured by Bayer in 1993, was indicated as antifibrinolytic to reduce blood loss during heart surgery35. By the end of 2007, aprotinin was discontinued globally, following Blood Conservation using Antifibrinolytics Trial (BART) findings that suggested an increase in 30-day mortality with aprotinin36. However, in 2012, the EMA recommended that the suspension be lifted37. However, these claims were disputed in another study in 2013, which found that aprotinin may increase the likelihood of mortality in low and intermediate cardiac surgery patients. The study suggested that the decision by the EMA to reinstate the drug for lower risk patients should be debated36.\n\n\nData mining in pharmacovigilance\n\nData mining is the process of collecting and analyzing data from sources of information that may be raw and complicated (such as data sets or databases) and extracting patterns of links and relationships between these data, to be translated into useful information38. Data mining has been used in many aspects; most importantly it has contributed to drug discoveries, prediction39, diagnosis of diseases (such as diabetes)40 in addition to drug complications and ADRs41. According to Wilson et al.42 “Data mining encompasses a number of statistical techniques including cluster analysis, link analysis, deviation detection and disproportionality assessment which can be utilized to determine the presence of and to assess the strength of ADR signals”. Whenever we predict these ADRs, we can reduce the morbidity and mortality rates43.\n\nMany studies have been published using data mining; for instance, in a data mining study to examine the relationship between antipsychotic drugs and myocarditis and cardiomyopathy using Bayesian statistics and found that myocarditis and cardiomyopathy were reported rarely as suspected ADRs, accounting for less than 0.1% (2121) of almost 2.5 million reports44. Furthermore, a study on benzodiazepines using data mining revealed the existence of potential signals for benzodiazepine-associated skin and subcutaneous tissue disorders45.\n\nUnfortunately, there are factors that affect the prediction of ADRs negatively. For instance, missing data is a major obstacle facing researchers worldwide, particularly with old cases. The issue of missing data is recognized, and several methods have been proposed and studied by researchers to fill the gap, like omitting records with missing information, or computerized modification of the data. However, these methods had their own limitations46. Underreporting of ADRs by healthcare professionals has a negative effect on data mining related to PV, especially for non-serious ADRs. Indeed, when the database is richer with reports and information related to “ordinary” ADRs, the mining process will be optimized; so that non-serious ADRs will act as a “background” against which critical ADRs will be prominent.\n\nDuplication in the reported cases, as well as duplicate information in the databases, are other impactful weakness in the data mining process.\n\nThe extracted information through data mining algorithms are not necessarily accurate and precise, so they must be evaluated clinically and dealt with cautiously before any decision is taken46.\n\nThe use of data mining in the PV of drugs has so far proven effective. Whether examining drugs with non-serious side effects or those that have been withdrawn from the market, data mining has been shown to be a valuable resource in the PV field47 and is currently implemented in the usual procedures of the major regulatory authorities and PV centers48.\n\nWith the growing popularity of social media globally, screening social networking sites are probably going to be a standard PV procedure. Therefore, utilization of data mining is likely to expand to mining social data for PV purposes49.\n\nThe aims of PV are well-recognized. It has been defined by the World Health Organization (WHO) as “the science and activities relating to the detection, assessment, understanding and prevention of adverse effects or any other drug-related problem”. The main focus of PV is to improve patient safety regarding the rational and safe use of medicine50. It also requires clinical staff to have competence in PV practices51. Furthermore, it is concerned with the early detection of unknown ADRs and identifying risk factors and causes of the ADRs49.\n\nPV, although a relatively new term that appeared in the seventies of the past century52, is not a new concept. In 1848, a 15-year-old female patient received chloroform as an anesthetic before treatment for an ingrown toenail. The patient developed ventricular fibrillation, which resulted in her death. In 1893, the Lancet published the results of a commission it established in Britain to report ADRs and deaths related to anesthesia. This is considered to be the prototype for an ADR spontaneous reporting system53.\n\nThe most notable event in PV appeared in 1961, when William Mc Bride, an Australian obstetrician, reported that in patients receiving thalidomide to treat morning sickness, up to a 20% surge in the development of fetal malformation was observed53. In the Netherlands, in a study by Lely in 1971, he reported the death of at least 19 people as a result of digitalis intoxication, which was due to an error in the production of the digitalis tablets54. Moreover, in 1974, after four years of marketing practolol in the UK, it was found to cause oculomucocutaneous syndrome and sclerosing peritonitis. Benoxaprofen was withdrawn from the market in 1982, only two years following marketing approval, due to multiple reports of photosensitivity and serious hepatotoxicity. These cases highlighted the importance of recording, reporting and publishing all ADRs. Such reports shed the light on less frequent ADRs and assist healthcare professionals in managing uncommon ADRs, which can be done by doing a quick database search for any similar cases55.\n\n\nPolicies and procedures for implementing pharmacovigilance\n\nThe WHO considers the monitoring of safety and effectiveness of medicines in any country as the responsibility of national governments. The fulfillment of this responsibility can be achieved by establishing national PV centers with well-defined PV systems. The foundation of the launch of PV centers is the establishment of policies regarding the costs, budget and the financial requirements of running the centers56. Financing for a PV center must be secured and given official approval, in order to guarantee the progress of work. The costs incurred depend on the size of population that is serviced by the center and the rate of reports generated. Possible sources of funding may be: insurance companies, academic institutions, and governmental bodies that are interested in the safety of medicinal products57. The location of the center may initially be within a country’s main hospital and, over time, be extended to multiple hospitals all over the country. Each smaller center would then send its reports to the main national center, which would be responsible for gathering the information and communicating and coordinating between the multiple centers. Finally, the main center would conduct and transfer the gathered information to the global PV institutions, such as Uppsala Monitoring Centre (UMC) in Sweden57.\n\nHowever, this will not be achieved unless the center has a sufficient number of qualified and trained personnel. The WHO determined that the minimum requirement of manpower to work in the PV center is at least one full-time employee58. Staff should give support and be involved in the PV process depending on their assigned tasks and responsibilities. However, these tasks and all organization resources should be structured and arranged to assist in the proper conduct of PV activities. Besides, the presence of qualified leadership is one of the most important factors in order to implement PV techniques and to motivate all staff to achieve the objectives. A good PV system must have excellent data collection methods to gather evidence on the risk/benefit balance and all criteria related to the safety of medicinal products, which will affect decision making. The system must include preparedness plans with appropriate instructions for urgent cases. The center should contain appropriate facilities and equipment which include office space and information technology systems59. Each center should have a database and a standard individual case safety report (ICSR) form and be connected with the national database. The national PV center should recruit an advisory committee, which will help and support the local PV centers in risk assessment and management, and most importantly in crisis58.\n\nAs the core of the PV system is to report any ADRs, reports must utilize a specific ADR reporting form and should be unique to each country. The forms should be distributed to all healthcare professionals in all areas, and all healthcare institutions; hospitals, pharmacies, medical centers60. These forms are known as standard ICSR forms58. ICSR is defined in PV as “a notification relating to a patient with an adverse medical event or laboratory test abnormality suspected to be induced by a medicine. It is an essential source of information for the achievement of the main objectives of PV and can involve several ADRs”61. Forms should then be gathered and collected or posted to the center by fax or email to ensure an easy flow of data. The minimum information in these forms should be general information about the patient (such as age, gender, race), a detailed description of the ADR, severity, lab tests (if any), date of appearance of ADR, the suspected medication causing the reported ADR (product information such as brand name, dosage form, the ingredients, and concentration), manufacturer related information, dose of medicine and date of initiation and withdrawal (if applicable), a brief medical history of the patient, the risk factors may be present in the patient like other medical problems or diseases (such as liver or renal problems and allergies), and the name of medical practitioner who detected and observed the ADR57.\n\nFor the reporting procedure to be complete, communication of ADR reports to VigiBase, the WHO global database that receives contributions from national PV centers in different countries, is essential for the success of the WHO’s International Drug Monitoring Programme56. The startup of the WHO’s Programme for International Drug Monitoring was in 1968 as a pilot project, with 10 countries already having established national systems for reporting of ADRs. The project then expanded to include more countries all over the world. New member countries developed PV centers to report the ADRs and coordinate with the WHO center in Uppsala, where VigiBase is based. VigiBase contains more than 8 million ADR reports from more than 110 countries62. VigiFlow is an internet-based system that offers free access to all member countries to see all information and reports in VigiBase, and their analysis from all over the world63. In April 2015, the WHO launched VigiAccess, a web application that allows anyone to access information. This is a significant step, which encourages reporting ADRs64.\n\nPV programs and drug regulatory authorities must be linked together so that the regulatory authority is continuously updated about any emerging safety issue, and at the same time regulatory authorities should know the critical need for the PV concept, leading to a focus not only on the approval of new medicines, but on their safety as well56.\n\nIn order to assure reliable PV program, it is crucial to periodically train staff on gathering and analyzing information related to ADRs, risk management, signal detection, data mining and possible actions in cases of a serious or fatal ADRs60. Today’s quick-paced advancing world implies that PV personnel may require training on new skills, technologies and concepts, like artificial intelligence and machine-learning.\n\nMoreover, training should be directed not only for the staff with specific PV tasks but also those with activities that have impact on the PV system, including clinical trials, and regulatory affairs59. As the training and education of healthcare professionals in the services of PV increases, patient safety will improve, quality of ADR reports will be enhanced, and the development of policies to prevent them will occur.\n\nA non-randomized study was conducted in a hospital in Brazil in 2012 on a multidisciplinary group of healthcare providers. Educational intervention involved different methods such as: lectures on PV, a practical class about reporting ADRs, and distribution of written materials about PV to healthcare providers. In order to assess the level of information about PV among the participants, a questionnaire was given before and after the intervention. The results showed that the educational intervention was successful in the understanding of PV concepts, and improving skills to effectively complete reports related to ADRs. Directly after the educational intervention, the number of reports increased; however, four months after the educational intervention the number decreased, which implies that the education has to be continuous or periodic to keep the motivation among healthcare practitioners toward reporting ADRs65.\n\n\nImplementation of pharmacovigilance regulations: country experiences\n\nAfter the most famous disaster of thalidomide in 1960's, the US FDA revised their regulations through imposing stricter rules on medicine approvals and establishing a spontaneous reporting PV system to report the ADR incidence in the healthcare sector53,66,67.\n\nA meta-analysis of prospective studies was performed from four electronic databases over a period of 32 years, from 1966 to 1996, including 39 studies in US hospitals in order to evaluate the occurrence of serious drug reactions. From these studies, it was found that a large number of hospital patients died from fatal ADRs, which were estimated to be the fourth to sixth leading cause of death in 1994, representing an important clinical issue66.\n\nThe FDA regulates PV with the help of the Center for Drug Evaluation and Research (CDER). This center evaluates new drugs before they can be marketed and maintains a rigorous post marketing safety surveillance program, monitoring the use of marketed drugs for unexpected health risks68.\n\nAccording to the American Society of Health-System Pharmacists (ASHP), ADR reports would vary due to the different size and type of the healthcare centers, patient mix, definition of ADR, and the medications used. The foremost obligation of reporting them is on pharmacists, physicians, nurses and even patients, and should include full information about the incident including the patient's name, patient's history etc. All ADR reports are analyzed and evaluated by a medical committee and in the case of serious ADR reports, they are reported to the FDA or drug's manufacturer or both69.\n\nMost serious ADR reporting, whether voluntary or mandatory, is done through the MEDWatch program, belonging to the FDA. This program was introduced in 199370. There are three forms that have been developed by the FDA for the reporting of ADRs: Form FDA 3500, for voluntary reporting by healthcare professionals; Form FDA 3500B, for voluntary reporting by patients and consumers; and Form FDA 3500A, for mandatory reporting71. The regulation and evaluation of ADRs may lead to regulatory action by the FDA, including labeling changes, risk management action plan (educating about the new safety information, and controlling distribution of the drug), removing the drug from the market, or conducting further studies72.\n\nA survey was conducted for a period of four months between May and August 2014 in three U.S. states (New Jersey, New York and Washington) in different health sectors, including pharmacists, nurses and physicians, to evaluate the ADR reporting process, and to explore gaps and issues in the reporting process. Results of the survey showed that the reporting process of ADRs was mainly to FDA MedWatch, internal reporting, and to the drug manufacturer. During the survey, factors for not reporting ADRs were: gaps in technology, gaps in education, and gaps in the overall process. Recommendations for improving the ADR reporting system include: improving integration between electronic systems, increasing awareness by training and educating patients and healthcare providers of the ADR reporting process, and simplifying and initiating a standard ADR-reporting process73.\n\nThe UK established an ADR monitoring system through spontaneous reporting, post marketing surveillance, and interrogating large databases. Spontaneous reporting of any ADR is carried out through the Yellow Card Scheme. Yellow Card reports are sent by healthcare professionals and patients by mail, telephone or through the internet to the Medicines and Healthcare product Regulatory Agency (MHRA). All reports are gathered and reviewed to detect any safety issue74.\n\nNewly marketed medicines are marked with an inverted black triangle. The triangle indicates that all doctors have to report all ADRs that are detected by patients after using the new medicine, through the Prescription-Event Monitoring technique. All reports are then submitted to the Committee on Safety of Medicines (CSM)75.\n\nThe French Agency for the Safety of Health Products, ANSM (Agence nationale de sécurité du médicament etdes produits de santé), is the authority responsible for PV activities implementation and coordination in France76.\n\nANSM succeeded AFSSAPS (Agence Française de Sécurité Sanitaire des Produits de Santé - French Agency for the Sanitary Safety of Health Products) as the PV regulation authority in 2011. AFSSAPS was heavily criticized for failing to withdraw Mediator (benfluorex) for a very long time, despite numerous reports of severe ADRs, and the fact that it was discontinued in several other countries. Therefore, the French government decided to establish ANSM in order to replace AFSSAPS14.\n\nANSM coordinates the national PV system, which is integrated into the European PV system. The PV system in France is based upon a network of 31 Regional PV Centers (Les centres régionaux de pharmacovigilance [CRPV]), responsible for monitoring, evaluating and preventing ADRs and risks, and promoting the optimum medicine usage. The CRPVs collect and transfer ADR reports to ANSM, and support healthcare professionals with information about PV. The Technical Pharmacovigilance Committee (Comité tech-nique de pharmacovigilance [CTPV]) comprises of all CRPV directors and ANSM management. CTPV is responsible for recommending “follow-up, analysis and prevention of risks”.\n\nHealthcare professionals may report ADRs to CRPV, or to the pharmaceutical company. Both CRPV and the pharmaceutical company should forward the report to ANSM. Patients now can report ADRs as well, through an online report form77.\n\nDue to the expansion of the expatriate population of the UAE, the healthcare system is attempting to meet mounting healthcare needs78. The main organizations responsible for the regulation of healthcare in the UAE are the Ministry of Health and Prevention (MOHAP), Department of Health – Abu Dhabi (DOH), and Dubai Health Authority (DHA).\n\nA number of policies and legislations have been drawn up regarding accessibility, availability, affordability, quality, and pricing of medicines; nevertheless, proper application remains a concern79. The UAE. initiated its PV program in 200879. It officially joined the WHO International Drug Monitoring Programme in collaboration with the Uppsala Monitoring Centre in 201380.\n\nStudies have been carried out to determine the knowledge, attitude, and practice (KAP) of ADR reporting among healthcare professionals in the UAE to identify their current strategies and pinpoint steps to reduce underreporting. One study found that there was poor KAP among healthcare providers in the UAE. The results showed that 81%, 83%, and 83.3% of doctors, community pharmacists, and hospital pharmacists, respectively, were unaware that the UAE had an ADR reporting center, and 56%, 60%, and 72% did not know the correct procedure for reporting ADRs. As such, it was noted that only 19%, 14%, and 12.1% of doctors, community pharmacists, and hospital pharmacists reported ADRs81. Further studies have shown that 72% of pharmacists and 86.7% of physicians were unaware of the ADR reporting system in UAE82.\n\n\nConclusions\n\nIn the past, PV has contributed to identifying the safety of medications at an earlier stage and thus preventing harmful effects of medicines affecting much larger populations. Several drugs have been banned based on the safety findings obtained during PV programs. Data mining is a powerful method of early detection of ADR signals and can provide valuable contribution to PV if properly integrated with modern information technology tools. Several countries have implemented policies governing PV that offered them substantial benefits in terms of ensuring patient safety.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "References\n\nMeyboom RH, Egberts AC, Gribnau FW, et al.: Pharmacovigilance in perspective. Drug Saf. 1999; 21(6): 429–447. PubMed Abstract \| Publisher Full Text\n\nPaludetto MN, Olivier-Abbal P, Montastruc JL: Is spontaneous reporting always the most important information supporting drug withdrawals for pharmacovigilance reasons in France? Pharmacoepidemiol Drug Saf. 2012; 21(12): 1289–1294. PubMed Abstract \| Publisher Full Text\n\nOlivier P, Montastruc JL: The nature of the scientific evidence leading to drug withdrawals for pharmacovigilance reasons in France. Pharmacoepidemiol Drug Saf. 2006; 15(11): 808–812. PubMed Abstract \| Publisher Full Text\n\nFoppiano M, Lombardo G: Worldwide pharmacovigilance systems and tolrestat withdrawal. Lancet. 1997; 349(9049): 399–400. PubMed Abstract \| Publisher Full Text\n\nTrifirò G, Pariente A, Coloma PM, et al.: Data mining on electronic health record databases for signal detection in pharmacovigilance: which events to monitor? Pharmacoepidemiol Drug Saf. 2009; 18(12): 1176–1184. PubMed Abstract \| Publisher Full Text\n\nSundaran S, Sankar S, Dhamodaran P, et al.: Drug recall: an overview. World J Pharm Res. 2013; 2: 297–307. Reference Source\n\nSibbald B: Rofecoxib (Vioxx) voluntarily withdrawn from market. CMAJ. 2004; 171(9): 1027–1028. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLenzer J: FDA is incapable of protecting US “against another Vioxx”. BMJ. 2004; 329(7477): 1253. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNichols DE: Dark classics in chemical neuroscience: lysergic acid diethylamide (LSD). ACS Chem Neurosci. 2018; 9(10): 2331–2343. PubMed Abstract \| Publisher Full Text\n\nCohen S, Dilman KS: Complications associated with lysergic acid diethylamide (LSD-25). JAMA. 1962; 181(2): 161–2. PubMed Abstract \| Publisher Full Text\n\nMurphy RC: A psychotherapist’s Debt to LSD. In Abramson, H. A., (ed) The use of LSD in psychotherapy and alcoholism, The Bobbs-Merrill Company, Inc. New York. 1967; 328.\n\nMullard A: Mediator scandal rocks French medical community. Lancet. 2011; 377(9769): 890–2. PubMed Abstract \| Publisher Full Text\n\nBoudemaghe T, Belhadj I: Data Resource Profile: The French National Uniform Hospital Discharge Data Set Database (PMSI). Int J Epidemiol. 2017; 46(2): 392–392d. PubMed Abstract \| Publisher Full Text\n\nBenkimoun P: New law introduces tougher rules on drug regulation in France. BMJ. 2011; 343: d8309. PubMed Abstract \| Publisher Full Text\n\nPamukcu Gunaydin G, Dogan NO, Levent S, et al.: Herbal weight loss pill overdose: sibutramine hidden in pepper pill. Case Rep Emer Med. 2015; 2015: 213874. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScheen AJ: Sibutramine on cardiovascular outcome. Diabetes Care. 2011; 34 Suppl 2: S114–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReuters: FDA pulls Parkinson's drug from U.S. market. Reference Source\n\nSafer DJ, Zito JM, Gardner JF: Pemoline hepatotoxicity and postmarketing surveillance. J Am Acad Child Adolesc Psychiatry. 2001; 40(6): 622–9. PubMed Abstract \| Publisher Full Text\n\nZito JM, Derivan AT, Kratochvil CJ, et al.: Off-label psychopharmacologic prescribing for children: history supports close clinical monitoring. Child Adolesc Psychiatry Ment Health. 2008; 2(1): 24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRidings JE: The thalidomide disaster, lessons from the past. Methods Mol Biol. 2013; 947: 575–86. PubMed Abstract \| Publisher Full Text\n\nLenz W: The Thalidomide hypothesis: how it was found and tested. In Epidemiological Concepts in Clinical Pharmacology. Springer, Berlin, Heidelberg. 1987; 3–10. Publisher Full Text\n\nTuccori M, Wallberg M: Other Sources of Information for Monitoring Drug Safety: Now and in the Future. In Pharmacovigilance. Adis, Cham. 2017; 181–193. Publisher Full Text\n\nCotter J, Wooltorton E: New restrictions on celecoxib (Celebrex) use and the withdrawal of valdecoxib (Bextra). CMAJ. 2005; 172(10): 1299. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNussmeier NA, Whelton AA, Brown MT, et al.: Complications of the COX-2 inhibitors parecoxib and valdecoxib after cardiac surgery. N Engl J Med. 2005; 352(11): 1081–91. PubMed Abstract \| Publisher Full Text\n\nAuffenberg C, Rosenthal LJ, Dresner N: Levamisole: a common cocaine adulterant with life-threatening side effects. Psychosomatics. 2013; 54(6): 590–3. PubMed Abstract \| Publisher Full Text\n\nLee KC, Ladizinski B, Federman DG: Complications associated with use of levamisole-contaminated cocaine: an emerging public health challenge. Mayo Clin Proc. Elsevier. 2012; 87(6): 581–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoorman-Li R, Motycka CA, Inge LD: A review of abuse-deterrent opioids for chronic nonmalignant pain. P T. 2012; 37(7): 412–8. PubMed Abstract \| Free Full Text\n\nShader RI: Dose dumping and the dumping of doses. J Clin Psychopharmacol. 2007; 27(4): 327–8. PubMed Abstract \| Publisher Full Text\n\nHenney JE: Withdrawal of troglitazone and cisapride. JAMA. 2000; 283(17): 2228. Publisher Full Text\n\nQuigley EM: Cisapride: what can we learn from the rise and fall of a prokinetic? J Dig Dis. 2011; 12(3): 147–56. PubMed Abstract \| Publisher Full Text\n\nBernard GR, Vincent JL, Laterre PF, et al.: Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med. 2001; 344(10): 699–709. PubMed Abstract \| Publisher Full Text\n\nCenter Watch: 2001 FDA Approved Drugs. 2017. Reference Source\n\nU.S. Food and Drug Administration: FDA Drug Safety Communication: Voluntary market withdrawal of Xigris [drotrecogin alfa (activated)] due to failure to show a survival benefit. 2011. Reference Source\n\nEuropean Medicines Agency: Xigris (drotrecogin alfa (activated)) to be withdrawn due to lack of efficacy. 2011. Reference Source\n\nTuffs A: Bayer withdraws heart surgery drug. BMJ. 2007; 335(7628): 1015. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeybohm P, Herrmann E, Nierhoff J, et al.: Aprotinin may increase mortality in low and intermediate risk but not in high risk cardiac surgical patients compared to tranexamic acid and ε-aminocaproic acid -- a meta-analysis of randomised and observational trials of over 30.000 patients. PLoS One. 2013; 8(3): e58009. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEuropean Medicines agency 2012 - aprotinin: European Medicines Agency, European Medicines Agency recommends lifting suspension of aprotinin. 2012. Reference Source\n\nMann RD, Andrews EB, editors: Pharmacovigilance. John Wiley & Sons; 2007. Reference Source\n\nRanjan J: Applications of data mining techniques in pharmaceutical industry. Journal of Theoretical & Applied Information Technology. 2007; 3(4). Reference Source\n\nSneha N, Gangil T: Analysis of diabetes mellitus for early prediction using optimal features selection. J Big Data. 2019; 6(1): 13. Publisher Full Text\n\nKarimi S, Wang C, Metke-Jimenez A, et al.: Text and data mining techniques in adverse drug reaction detection. ACM Comput Surv. 2015; 47(4): 56. Publisher Full Text\n\nWilson AM, Thabane L, Holbrook A: Application of data mining techniques in pharmacovigilance. Br J Clin Pharmacol. 2004; 57(2): 127–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCami A, Arnold A, Manzi S, et al.: Predicting adverse drug events using pharmacological network models. Sci Transl Med. 2011; 3(114): 114ra127. PubMed Abstract \| Publisher Full Text\n\nCoulter DM, Bate A, Meyboom RH, et al.: Antipsychotic drugs and heart muscle disorder in international pharmacovigilance: data mining study. BMJ. 2001; 322(7296): 1207–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVega BP, Bueso JJ, Arroyo PA, et al.: Data mining: Pharmacovigilance signal of benzodiazepines and skin and subcutaneous tissue disorders. PS026. BMJ. 2016; 23(Suppl 1). Publisher Full Text\n\nPoluzzi E, Raschi E, Piccinni C, et al.: Data mining techniques in pharmacovigilance: analysis of the publicly accessible FDA adverse event reporting system (AERS). In Data mining applications in engineering and medicine. IntechOpen. 2012. Publisher Full Text\n\nDipali B, Yogita T: Data mining as a tool for detecting adverse effects of drugs. International Conference on Computing, Communication and Automation (ICCCA). 2016. Publisher Full Text\n\nHauben M, Madigan D, Gerrits CM, et al.: The role of data mining in pharmacovigilance. Expert Opin Drug Saf. 2005; 4(5): 929–48. PubMed Abstract \| Publisher Full Text\n\nPappa D, Stergioulas LK: Harnessing social media data for pharmacovigilance: a review of current state of the art, challenges and future directions. Int J Data Sci Anal. 2019; 8(2): 1–23. Publisher Full Text\n\nWorld Health Organization: Pharmacovigilance.2017, [Accessed 4 April. 2017]. Reference Source\n\nReumerman M, Tichelaar J, Piersma B, et al.: Urgent need to modernize pharmacovigilance education in healthcare curricula: review of the literature. Eur J Clin Pharmacol. 2018; 74(10): 1235–48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeninger P: Pharmacovigilance: an overview. Clin Ther. 2018; 40(12): 1991–2004. PubMed Abstract \| Publisher Full Text\n\nRoutledge P: 150 years of pharmacovigilance. Lancet. 1988; 351(9110): 1200–1201. PubMed Abstract \| Publisher Full Text\n\nVan Grootheest K: The dawn of pharmacovigilance. Int J Pharm Med. 2003; 17(5–6): 195–200. Publisher Full Text\n\nShah RR: Importance of publishing adverse drug reaction case reports: promoting public health and advancing pharmacology and therapeutics. Drug Saf Case Rep. 2017; 4(1): 11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization Policy Perspectives on Medicines: Pharmacovigilance: ensuring the safe use of medicines. Geneva. 2004. Reference Source\n\nChakrabarty M, Thawani V: Starting a pharmacovigilance center: actions for implementation. J Pharmacol Pharmacother. 2011; 2(4): 295–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO 2010 World Health Organization: Minimum Requirements for a Functional Pharmacovigilance System. Geneva.2010. Reference Source\n\nEuropean Medicines Agency: Guideline on good Pharmacovigilance practices (GVP), module I Pharmacovigilance systems and their quality systems. 2012. Reference Source\n\nBiswas P, Biswas AK: Setting standards for proactive pharmacovigilance in India: The way forward. Ind J Pharmacol. 2007; 39(3): 124–8. Publisher Full Text\n\nAgu KA, Oparah AC: Adverse drug reactions to antiretroviral therapy: Results from spontaneous reporting system in Nigeria. Perspect Clin Res. 2013; 4(2): 117–24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nViola E, Opri S, Moretti U, et al.: α1-Adrenergic receptor antagonists and gynecomastia. A case series from the Italian spontaneous reporting system and VigiBase(™). Eur J Clin Pharmacol. 2014; 70(8): 1003–9. PubMed Abstract \| Publisher Full Text\n\nOlsson S, Pal SN, Dodoo APharmacovigilance in resource-limited countries. Exp Rev Clin Pharmacol. 2015; 8(4): 449–460. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: WHO essential medicines and health products: annual report 2015. World Health Organization. 2016. Reference Source\n\nVarallo FR, Planeta CS, Mastroianni PD: Effectiveness of pharmacovigilance: multifaceted educational intervention related to the knowledge, skills and attitudes of multidisciplinary hospital staff. Clinics (Sao Paulo). 2017; 72(1): 51–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLazarou J, Pomeranz BH, Corey PN: Incidence of adverse drug reactions in hospitalized patients: a meta-analysis of prospective studies. JAMA. 1998; 279(15): 1200–5. PubMed Abstract \| Publisher Full Text\n\nFeibus KB: FDA’s proposed rule for pregnancy and lactation labeling: improving maternal child health through well-informed medicine use. J Med Toxicol. 2008; 4(4): 284–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarpenter DP: The political economy of FDA drug review: processing, politics, and lessons for policy. Health Aff (Millwood). 2004; 23(1): 52–63. PubMed Abstract \| Publisher Full Text\n\nAmerican Society of Health-System Pharmacists: ASHP guidelines on adverse drug reaction monitoring and reporting. American Society of Hospital Pharmacy. Am J Health Syst Pharm. 1995; 52(4): 417–9. PubMed Abstract \| Publisher Full Text\n\nJohnson C: Software tools to support incident reporting in safety-critical systems. Saf Sci. 2002; 40(9): 765–780. Publisher Full Text\n\nKhoie T, Zinderman CE, Solomon R, et al.: Clarification of FDA and The Joint Commission reporting requirements for US tissue recipient adverse reactions. Cell Tissue Bank. 2008; 9(1): 67–8. PubMed Abstract \| Publisher Full Text\n\nGad SC: Drug safety evaluation. John Wiley & Sons; 2016. Publisher Full Text\n\nStergiopoulos S, Brown CA, Felix T, et al.: A Survey of Adverse Event Reporting Practices Among US Healthcare Professionals. Drug Saf. 2016; 39(11): 1117–1127. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAvery AJ, Anderson C, Bond CM, et al.: Evaluation of patient reporting of adverse drug reactions to the UK ‘Yellow Card Scheme’: literature review, descriptive and qualitative analyses, and questionnaire surveys. Health Technol Assess. 2011; 15(20): 1–234, iii-iv. PubMed Abstract \| Publisher Full Text\n\nHeeley E, Riley J, Layton D, et al.: Prescription-event monitoring and reporting of adverse drug reactions. Lancet. 2001; 358(9296): 1872–3. PubMed Abstract \| Publisher Full Text\n\nMoore N, Kreft-Jais C, Dhanani A: Spontaneous reporting–France. In: Mann RD, Andrews EB, eds. Pharmacovigilance. 2007. Publisher Full Text\n\nVial T: French pharmacovigilance: missions, organization and perspectives. Therapie. 2016; 71(2): 143–50. PubMed Abstract \| Publisher Full Text\n\nHassan R, Sher HA, Khokhar R, et al.: Pharmaceutical Policy in the UAE. In: Pharmaceutical Policy in Countries with Developing Healthcare Systems. Adis, Cham. 2017; 365–379. Publisher Full Text\n\nWilbur K: Pharmacovigilance in the middle east. Drug Saf. 2013; 36(1): 25–30. Publisher Full Text\n\nDameh M: United Arab Emirates pharmacists’ practices and views on adverse drug reaction and medication error reporting and health professional expectations. IDSR-JPBS. 2015; 10(1): 86–96. Reference Source\n\nSaid ASA, Hussain N: Adverse Drug Reaction Reporting Practices Among United Arab Emirates Pharmacists and Prescribers. Hosp Pharm. 2017; 52(5): 361–366. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSathvik BS, Chukir DM, Abo-Aldan E, et al.: Adverse drug reaction monitoring and reporting: knowledge, attitude and belief of physicians & pharmacists of Ras Al Khaimah, United Arab Emirates (UAE). Int J Pharm Res. 2014; 5(2): 368–375. Publisher Full Text" }	[ { "id": "57875", "date": "23 Dec 2019", "name": "Nisha Jha", "expertise": [ "Reviewer Expertise Pharmacovigilance" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article has been written in a very appropriate way. This review article has also used relevant literature and the information given is very appropriate in the field of pharmacovigilance. The references are used accurately. Data miming information about the pharmacovigilance has been considered as an important area of information and this article will help young researchers to work more in this area. Overall, the article has been very well written.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] }, { "id": "60295", "date": "28 Feb 2020", "name": "Mohammed Alshakka", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAn excellent article in which the authors exerted a great and wonderful effort and discussed an extremely important topic for HCP, patients and health in general:\nI have only two comments which will strengthen the article:\nPlease add to your text example for low income countries and add several challenges that face running strong pharmacovigilance activities in developing countries.\n\nWhat are the strategies to uplift the current PV program in UAE and developing countries? Strategies such as teaching PV and enhancing patients and consumer reporting etc...\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-2109
https://f1000research.com/articles/8-2108/v1	16 Dec 19	{ "type": "Research Article", "title": "Nanopore long reads enable the first complete genome assembly of a Malaysian Vibrio parahaemolyticus isolate bearing the pVa plasmid associated with acute hepatopancreatic necrosis disease", "authors": [ "Han Ming Gan", "Christopher M Austin", "Christopher M Austin" ], "abstract": "Background: The genome of Vibrio parahaemolyticus MVP1, isolated from a Malaysian aquaculture farm with shrimp acute hepatopancreatic necrosis disease (AHPND), was previously sequenced using Illumina MiSeq and assembled de novo, producing a relatively fragmented assembly. Despite identifying the binary toxin genes in the MVP1 draft genome that were linked to AHPND, the toxin genes were localized on a very small contig precluding proper analysis of gene neighbourhood. Methods: The genome of MVP1 was sequenced on Nanopore MinION to obtain long reads to improve genome contiguity. De novo genome assembly was performed using long-read only assembler followed by genome polishing and hybrid assembler. Results: Long-read assembly produced three complete circular MVP1 contigs: chromosome 1, chromosome 2 and the pVa plasmid encoding pirABvp binary toxin genes. Polishing of the long-read assembly with Illumina short reads was necessary to remove indel errors. Complete assembly of the pVa plasmid could not be achieved using Illumina reads due to identical repetitive elements flanking the binary toxin genes leading to multiple contigs. These regions were fully spanned by the Nanopore long-reads resulting in a single contig. Alignment of Illumina reads to the complete genome assembly indicated there is sequencing bias as read depth was lowest in low-GC genomic regions. Comparative genomic analysis revealed a gene cluster coding for additional insecticidal toxins in chromosome 2 of MVP1 that may further contribute to host pathogenesis pending functional validation. Scanning of publicly available V. parahaemolyticus genomes revealed the presence of a single AinS-family quorum-sensing system that can be targeted for future microbial management. Conclusions: We generated the first chromosome-scale genome assembly of a Malaysian pirABVp-bearing V. parahaemolyticus isolate. Structural variations identified from comparative genomic analysis provide new insights into the genomic features of V. parahaemolyticus MVP1 that may be associated with host colonization and pathogenicity.", "keywords": [ "Vibrio parahaemolyticus", "Nanopore", "Illumina", "quorum-sensing", "shrimp", "Acute Hepatopancreatic Necrosis Disease" ], "content": "Introduction\n\nVibrio parahaemolyticus is a marine gram-negative Enterobacteriaceae that has been recognized as an important pathogen affecting commercially-relevant shrimp species such as the giant tiger prawn (Penaeus monodon) and the Pacific white shrimp (Litopenaeus vannamei)1–3. In recent years, V. parahaemolyticus has been linked to acute hepatopancreatic necrosis disease (AHPND)4,5. An AHPND-causing V. parahaemolyticus isolate is thought to produce a homolog of the insecticidal Photorhabdus insect-related (Pir) binary toxin that induces prolonged damage to the shrimp tissue2. AHPND preferentially affects shrimp post-larvae or juveniles with nearly 100% mortality rate within 30–35 days post-stocking6. Studies suggest that AHPND originated from China in 2009 and subsequently spread to South East Asia7. In Malaysia, AHPND was first reported in 2011 where it still persists in shrimp farms8.\n\nThe implementation of effective microbial management in aquaculture requires genomic surveillance data that can provide accurate information regarding the geographical origin, virulence factors and antibiotic resistance of a sequenced microbial strain9. In Malaysia, this remains challenging due to the paucity of available genomic resources for the relevant pathogens. To date, a majority of the microbial genomic studies in Malaysia consist of single draft genome reports mostly focusing on clinical pathogens without substantial comparative genomics10–13. However, recent years have seen a modest increase in the number of studies with more comprehensive sequencing dataset and analysis14–16. For example, Yan et al. sequenced and assembled the draft genomes of 40 Malaysian V. parahaemolyticus isolates associated with shrimp aquaculture and performed comparative genomics of more than Asian 100 V. parahaemolyticus genomes from public databases. In-silico multi-locus sequencing typing and phylogenomic analyses indicate that several Malaysian V. parahaemolyticus isolates belong to previously undescribed sequence types and genomic lineages17 and recommended further studies be undertaken.\n\nThe pirABvp genes coding for AHPND-causing binary toxins are localized on a 70-kb pVa plasmid2. Surprisingly, despite the small size of the plasmid relative to the chromosomal genome, a complete de novo assembly of the pVa plasmid using Illumina-only short reads has not been achieved to date. Recent studies suggested that this is due to the presence of repetitive elements on the pVa plasmid that are longer than the Illumina read length18. As a result, pirAB-containing contigs that were assembled from Illumina reads are generally short with limited gene content, precluding detailed analysis of the pirAB gene neighborhood and their stability in the plasmidome19. V. parahaemolyticus isolate MVP1 is one of the first pirABVp -harboring V. parahaemolyticus isolated from Malaysia to have its genome sequenced11. Isolate MVP1 was previously sequenced on an Illumina MiSeq followed by de novo genome assembly using the SPAdes assembler. While the pirABvp genes could be identified in the draft genome, the assembly was problematic as these were localized on a short contig flanked by small fragments of a transposase gene.\n\nRecent years have seen the democratization of long-read sequencing enabled by Nanopore technology. In contrast to another popular long-read sequencing platform, PacBio, Nanopore sequencing requires minimal lab footprint and very low capital investment. Importantly, it is the first sequencing technology that allows native sequencing, thus eliminating sequencing bias associated with the activity of Taq-polymerase. Long reads have been utilized for de novo genome assembly in two distinct ways. First, long reads can be used directly in genome assembly followed by polishing with Illumina short reads to improve consensus accuracy20. An alternative approach utilizes long-reads to reorder and link contigs that were initially assembled from Illumina reads21,22. While the final assembly produced with this hybrid approach is highly accurate, the contiguity of the assembly is dependent on the quality of the initial Illumina assembly23.\n\nLeveraging on the availability of Illumina dataset for isolate MVP1, we aimed to improve on the original genome assembly11 through the addition of 40× Nanopore read coverage. We first performed a Nanopore-led assembly using the long-read Flye assembler24 followed by polishing with Illumina reads. In addition, we generated a hybrid assembly using Unicycler assembler23. Although both methods produced a complete circular pVa plasmid sequence with intact pirABvp, complete chromosome assembly was only achieved with the Nanopore-led Flye assembly. This superior assembly provides new insight into the genomic features of V. parahaemolyticus MVP1 and will allow a greater understanding of host pathogenicity through comparative genomic analysis with selected V. parahaemolyticus isolates.\n\n\nMethods\n\nSample collection, gDNA isolation and Illumina sequencing of V. parahaemolyticus isolate MVP1 have been previously reported11. For Nanopore sequencing, 1 μg of unfragmented gDNA was processed using the now obsolete Nanopore SQK-NSK007 library preparation kit. Sequencing was subsequently performed on an R9 flowcell attached to a MinION device for 48 hours. Base-calling of the produced raw fast5 files used Guppy v3.1 (high accuracy mode; requires registration to Oxford Nanopore Technology community site).\n\nIllumina-only assembly and hybrid assembly incorporating high-quality (q>7) Nanopore long reads were performed using Unicycler v.0.4.723. Only contigs equal or larger than 1,000 bp were retained for subsequent analyses. A long-read only de novo assembly was also performed with Flye v2.4.2 utilizing similar Nanopore sequencing data24. Illumina polishing of the Flye assembly used the unicycler polish tool in Unicycler v.0.4.7 that performed multiple rounds of bwa-mem-based Illumina read alignment to the assembly followed by polishing with Pilon v1.2225. Genome completeness was assessed with BUSCO v3 (Gammaproteobacteria odb9) based on the whole proteome from each genome assembly that was predicted using Prodigal v 2.6.3 (default setting)26,27.\n\nAutomated annotation of the whole-genome was carried out using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP)28. The translated coding sequences generated from PGAP were subject to additional functional annotation using InterProScan. Intra-chromosomal structural variations and sequence similarity were visualized with BLAST ring image generator (BRIG) using the blastN (e-value = 0.001 cutoff). Sequencing depth was also calculated in BRIG based on the BAM alignment file generated from the bwa-mem mapping of Illumina paired-end reads to the whole-genome29. Structural modeling of the putative insecticidal toxin proteins identified in MVP1 used the online Phyre2 webserver30.\n\nVibrio proteins containing the InterPro signature IPR035304, corresponding to the AinS-family N-acyl-homoserine-lactone autoinducer synthase, were downloaded from the UniProt database31 on the 7th of November 2019 and clustered at 98% amino acid identity cutoff using cd-hit v4.632. The protein clusters were subsequently aligned with MAFFT v7.3133 (”--maxiterate 1000 –localpair” setting) followed by maximum likelihood tree construction using FastTree v2.134. Calculation and visualization of pairwise protein sequence divergence used SDT v1.235. In addition, to confirm the absence of the LuxI-type autoinducer synthase homolog in the Vibrio parahaemolyticus proteomes, additional domain search specifically for PF00765 (LuxI autoinducer synthase domain) was performed followed by subsequent InterProScan validation as previously described36.\n\n\nResults and discussion\n\nA total of 231 megabases of Nanopore data contained in 30,315 reads (N50 = 10,908 bp) were produced from a single Nanopore MINION run on the discontinued R9 flowcell. The base-called Nanopore fastq file has been deposited in the SRA database under the accession code SRX6759854. An initial Unicycler assembly using previously generated Illumina reads (SRX6759853) yielded a relatively fragmented genome (198 contigs; N50 length = 54,109 bp). Supplementing the assembler with approximately 40 x coverage of Nanopore long reads led a substantial improvement in the Unicycler assembly contiguity (9 contigs; N50 length = 1,738,848 bp). Of the 9 assembled contigs, one of them was flagged as “complete” with a contig length of 70kb. On the other hand, the Flye assembly using only Nanopore reads produced three contigs all flagged as “complete and circular”. Despite generating the most contiguous assembly, the initial Flye assembly has the worst BUSCO score with 37.4% BUSCO single-copy and 45.1% fragmented BUSCO genes (Table 1). Polishing of the Flye assembly with Illumina paired-end reads restored the genome completeness to a level that is comparable to the hybrid and Illumina-only assemblies (Table 1 and Extended data: Supplemental File 1). The high percentage of fragmented genes in long-read-only assembly is usually a result of erroneous frameshifts from indel sequencing errors in the long reads20. Although higher coverage of long read may lead to an increase the consensus accuracy; its accuracy will unlikely match that of an Illumina-polished assembly due to Nanopore-specific systematic errors. As a result, polishing of long-read assembly using Illumina short reads can be considered as a cost-effective but less convenient approach for producing highly accurate and contiguous microbial genome assembly.\n\nThe alignment of Illumina reads to the final chromosomal assembly revealed substantial lower sequencing depths in genomic regions with low GC-content (<30%) (Figure 1). Some of these regions are unique to MVP1 and may risk being overlooked in the Illumina-only assembly at lower sequencing coverage. For example, less than 5x sequencing depth was observed in the region 3,240 to 3,280 kb that contain various putative carbohydrate metabolism genes that are unique to isolate MVP1 in this current genomic comparison. The incorporation of PCR during library preparation is known to reduce the representation of high-AT genomic regions due to the polymerase amplification bias towards GC-balanced fragments37. Reduced Illumina sequencing depth in high-AT genomic regions affecting biological interpretation has also been reported in several crustacean mitogenomic libraries as both the phylogenetically informative control region and 16S rRNA genes are relatively AT-rich38,39. Depending on the amount of DNA available for sequencing, a few modifications to the Illumina library preparation step can be considered, such as PCR-free preparation for sample with high starting DNA (> 100 ng) and reduced PCR cycle coupled with higher (>100× genome coverage) sequencing depth for samples with low starting DNA.\n\nLeft, V. parahaemolyticus MVP1 chromosome 1 compared against five other V. parahaemolyticus chromosome 1. Right, V. parahaemolyticus MVP1 chromosome 2 compared against five other V. parahaemolyticus chromosome 2. The innermost rings show GC content while the second innermost rings show Illumina sequencing depth along the genome (blue). The remaining rings indicate BLAST comparisons of other V. parahaemolyticus genomes against MVP genome assembly. Arrows indicate genes of interest and were labeled with the predicted protein names.\n\nIn the Illumina-only assembly, the pVa plasmid was assembled into three contigs (Contig24: 63.4 kb, Contig164: 3.4kb; Contig198: 1 kb). Contig198 contains a 921-bp gene that codes for an intact IS5 family transposase and could be aligned equally well to two genomic regions flanking the pirABVp genes in the complete plasmid assembly (Figure 2). This observation can be explained by the inability of Illumina reads to fully span the repeats thus causing them to be collapsed into a single contig. As a result, the localization of pirABVp on the pVa plasmid cannot be confirmed from an Illumina-only assembly. In contrast the entire pirABVp genes and their flanking transposase genes were fully covered by a number of Nanopore long reads enabling the complete assembly of the pVa plasmid in addition to supporting the localization of pirABVp genes on the pVa plasmid. Nanopore read depth compared to Illumina, is relatively even across the aligned genomic region, which is consistent with the absence of PCR bias in the Nanopore library preparation.\n\nDirection of arrow in the annotation indicates transcription orientation. Blue and red arrows in the Nanopore read alignment indicate forward and reverse strands, respectively.\n\nThe minor structural variations previously reported in pirABVp -containing region suggest that these genes are not stably maintained and are prone to transposition mediated by the flanking IS5 family transposase genes40. Interestingly, the localization of this IS5 family transposase gene is not specific to just the pVa plasmid. Local nucleotide similarity search of this transposase gene against the complete MVP genome revealed three additional perfect hits (100% query coverage and 100% nucleotide identity) in each of the chromosomal genome (Figure 1). The localization of these additional IS5 family transposase in both chromosome 1 and chromosome 2 further raises the possibility of the IS5 transposase-flanked pirABVp genes being integrated into the chromosomal region41,42.\n\nChromosome 2 of Isolate MPV1 consists of a 160 kb genomic region that is mostly absent in five out of six pVa plasmid-harboring V. parahaemolyticus isolates included in this comparative genomic analysis. Functional annotation of the genes located in this region revealed two gene clusters that may further contribute to host pathogenicity in addition to the binary Pir-like toxins on the pVa plasmid. The first gene cluster located from 1,822,499 to 1,836,895 bp in chromosome 2 consists of three relatively large genes (Gene Locus Tag: BSR23_26145, BSR23_26150 and BSR23_26155) coding for another type of putative insecticidal toxins. Functional annotation using InterProScan revealed the presence of protein domains commonly associated with the toxin-complex (Tc) toxins (Figure 3). In addition, Phyre2 protein modeling also indicated their high structural homology to their respective homologous toxin components (Extended data: Supplemental Files 2–4). In Xenorhabdus nematophilus, the three toxin components formed a native toxin complex and the ingestion of this complex led to growth inhibition of two different insect larvae, Helicoverpa zea and Heliothis virescens43. In addition, further tests showed that the native toxin complex is capable of binding to solubilized gut membranes of H. zea larvae in addition to inducing pore formation in black lipid membranes43. Given the functional resemblance of such toxins to the Pir-like binary toxins, the presence of all three genes coding for the complete set of insecticidal Tc toxins in V. parahaemolyticus may contribute to mortality without AHPND lesions in shrimps, as previously reported44.\n\nInterestingly, isolate MVP1 also harbor a fuc operon that is not commonly present in members of this species (Extended data: Supplemental Table 1). The presence of this operon in MVP1 translates into the genomic potential for the internalization of fucose for incorporation into the capsular polysaccharides or bacterial glycoproteins. While it is also possible that the fuc operon may contribute to improved utilization of fucose as an energy source, Williams et al. did not observe a greater capacity to utilize fucose in an EMS-causing V. parahaemolyticus isolate 13-028/A345.\n\nNCBI annotation of MVP1 proteome revealed the presence of only the LuxM/OpaM-type AHL synthase. An in-house HMMsearch for the LuxI autoinducer synthase domain (PFAM signature: PF00765) in the MVP1 proteome also did not reveal significant hits, indicating that MVP1 employs a single system for the biosynthesis of AHL signal (Extended Data: Supplemental File 5). Additional UniProt query search of “PF00765 AND Vibrio parahaemolyticus” on 14th November 2019, showed only one positive hit across all currently available V. parahaemolyticus proteomes. However, further investigation of the positive hit, AAY51_09590, revealed that this belonged to a Citrobacter spp. previously misclassified as V. parahaemolyticus46. In contrast, V. harveyi was shown to utilize at least two quorum-sensing systems e.g. LuxI that synthesizes 3-oxo-C6-HSL and the AinS that synthesizes C8-HSL47. Despite being classified as members of the same family for sharing the similar protein domain signature, AinS exhibited a substantially lower pairwise amino acid divergence (~ 30%) in comparison to LuxM and OpaM. By rooting the LuxM/OpaM phylogenetic tree with the AinS from Aliivibrio fischeri as the outgroup, we observed a strongly supported monophyletic cluster consisting of the functionally validated LuxM and OpaM in addition to their homologs from other Vibrio spp (Figure 4A). The pairwise amino acid identity among members in this LuxM/OpaM clade averages around 50% (Figure 4B), consistent with the relatively short branch length among members of the clade.\n\n(A) Maximum likelihood tree depicting the evolutionary relationships of AinS-like family protein clusters among Vibrio spp. Number after the “=” sign in each tip label indicates the number of proteins represented by the cluster. Node were colored according to the SH-like local support values and branch lengths indicate number of substitutions per site. (B) Pairwise identity matrix of the identified AinS-like family proteins in Vibrio spp.\n\nIn Indian white shrimps (Fenneropenaeus indicus), co-injection of a pathogenic V. parahaemolyticus strain DHAP1 and purified recombinant AHL lactonase, AiiA, reduced Vibrio viable counts and biofilm development in the shrimp intestine, suggesting the role of AHL-mediated quorum-sensing system in host colonization48. While the presence of intact opaM in several sequenced V. parahaemolyticus isolates supports their ability to accumulate AHL and engage in quorum-sensing activity, this is best validated using a simple biosensor or LC/MS approach that can not only verify the AHL producing phenotype but also provide insight into the structural diversity of AHL signals49–51. Furthermore, elucidating the role of opaM in mediating host colonization and pathogenesis among AHPND-causing V. parahaemolyticus via transposon mutagenesis will be instructive.\n\n\nConclusions\n\nUsing approximately 40× genome coverage of Nanopore long reads, we produced the first chromosome-scale genome assembly of a Malaysian pirABVp-bearing V. parahaemolyticus isolate. Although the genome completeness of the initial Flye assembly was relatively poor, polishing of the genome assembly with Illumina reads improved its completeness to a level that is comparable to a high-quality microbial genome assembly. Structural variations identified from genomic comparisons provide new insights into the genomic features of V. parahaemolyticus MVP1 that may be associated with host colonization and pathogenicity.\n\n\nData availability\n\nVibrio parahaemolyticus strain MVP1 chromosome 1, complete sequence, Accession number CP043421: https://www.ncbi.nlm.nih.gov/nuccore/CP043421\n\nVibrio parahaemolyticus strain MVP1 chromosome 2, complete sequence, Accession number CP043422: https://www.ncbi.nlm.nih.gov/nuccore/CP043422\n\nVibrio parahaemolyticus strain MVP1 plasmid pVa, complete sequence, Accession number CP043423: https://www.ncbi.nlm.nih.gov/nuccore/CP043423\n\nRaw Illumina reads and basecalled Nanopore Reads have also been deposited in NCBI Sequence Read Archive under the BioProject PRJNA355061.\n\nZenodo: Nanopore long reads enable the first complete genome assembly of a Malaysian Vibrio parahaemolyticus isolate bearing the pVa plasmid associated with acute hepatopancreatic necrosis disease, http://doi.org/10.5281/zenodo.356848552.\n\nThis project contains the following extended data:\n\nSupplemental File 1: Main genome assemblies (Unpolished Flye assembly, Polished Flye assembly, Unicycler Hybrid Assembly and Unicycler Illumina-only assembly) generated in this study for comparison and their BUSCO output.\n\nSupplemental File 2: Phyre2 protein modeling output of the putative MVP1 TcdA toxin\n\nSupplemental File 3: Phyre2 protein modeling output of the putative MVP1 TcdB toxin\n\nSupplemental File 4: Phyre2 protein modeling output of the putative MVP1 TccC toxin\n\nSupplemental File 5: InterProScan output of the NCBI-predicted MVP1 proteome.\n\nSupplemental Table 1: NCBI BlastN output using the fuc genes of Vibrio parahaemolyticus MVP1 as the query to search against the Vibrio reference WGS database as of 21 Oct 2019.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nHaldar S, Chatterjee S, Asakura M, et al.: Isolation of Vibrio parahaemolyticus and Vibrio cholerae (Non-O1 and O139) from moribund shrimp (Penaeus monodon) and experimental challenge study against post larvae and juveniles. Ann Microbiol. 2007; 57(1): 55–60. Publisher Full Text\n\nLee CT, Chen IT, Yang YT, et al.: The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin. Proc Natl Acad Sci U S A. 2015; 112(34): 10798–803. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYaashikaa PR, Saravanan A, Kumar PS: Isolation and identification of Vibrio cholerae and Vibrio parahaemolyticus from prawn (Penaeus monodon) seafood: Preservation strategies. Microb Pathog. 2016; 99: 5–13. PubMed Abstract \| Publisher Full Text\n\nKhimmakthong U, Sukkarun P: The spread of Vibrio parahaemolyticus in tissues of the Pacific white shrimp Litopenaeus vannamei analyzed by PCR and histopathology. Microb Pathog. 2017; 113: 107–12. PubMed Abstract \| Publisher Full Text\n\nSoto-Rodriguez SA, Gomez-Gil B, Lozano-Olvera R, et al.: Field and experimental evidence of Vibrio parahaemolyticus as the causative agent of acute hepatopancreatic necrosis disease of cultured shrimp (Litopenaeus vannamei) in Northwestern Mexico. Appl Environ Microbiol. 2015; 81(5): 1689–99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHong XP, Xu D, Zhuo Y, et al.: Identification and pathogenicity of Vibrio parahaemolyticus isolates and immune responses of Penaeus (Litopenaeus) vannamei (Boone). J Fish Dis. 2016; 39(9): 1085–97. PubMed Abstract \| Publisher Full Text\n\nHong X, Lu L, Xu D: Progress in research on acute hepatopancreatic necrosis disease (AHPND). Aquac Int. 2016; 24(2): 577–93. Publisher Full Text\n\nKua BC, Iar A, Siti Zahrah A, et al.: Current status of acute hepatopancreatic necrosis disease (AHPND) of farmed shrimp in Malaysia. Addressing Acute Hepatopancreatic Necrosis Disease (AHPND) and Other Transboundary Diseases for Improved Aquatic Animal Health in Southeast Asia: Proceedings of the ASEAN Regional Technical Consultation on EMS/AHPND and Other Transboundary Diseases for Improved Aquatic Animal Health in Southeast Asia, 22–24 February 2016, Makati City, Philippines; Aquaculture Department, Southeast Asian Fisheries Development Center. 2016. Reference Source\n\nRelman DA: Microbial genomics and infectious diseases. N Engl J Med. 2011; 365(4): 347–57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYap KP, Gan HM, Teh CS, et al.: Genome Sequence and Comparative Pathogenomics Analysis of a Salmonella enterica Serovar Typhi Strain Associated with a Typhoid Carrier in Malaysia. J Bacteriol. 2012; 194(21): 5970–1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFoo SM, Eng WWH, Lee YP, et al.: New sequence types of Vibrio parahaemolyticus isolated from a Malaysian aquaculture pond, as revealed by whole-genome sequencing. Genome Announc. 2017; 5(19): pii: e00302-17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOsama A, Gan HM, Teh CS, et al.: Genome sequence and comparative genomics analysis of a Vibrio cholerae O1 strain isolated from a cholera patient in Malaysia. J Bacteriol. 2012; 194(24): 6933. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGan HM, Rajasekaram G, Eng WWH, et al.: Whole-Genome Sequences of Two Carbapenem-Resistant Klebsiella quasipneumoniae Strains Isolated from a Tertiary Hospital in Johor, Malaysia. Genome Announc. 2017; 5(32): pii: e00768-17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGan HM, Eng WWH, Dhanoa A: Data on whole-genome sequencing of extended-spectrum beta-lactamases producing Enterobacteriaceae isolates from Malaysia. Data Brief. 2019; 25: 104257. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGan HM, Eng WWH, Dhanoa A: First genomic insights into carbapenem-resistant Klebsiella pneumoniae from Malaysia. J Glob Antimicrob Resist. 2019; pii: S2213-7165(19)30174-2. PubMed Abstract \| Publisher Full Text\n\nYap KP, Ho WS, Gan HM, et al.: Global MLST of Salmonella Typhi revisited in post-genomic era: genetic conservation, population structure, and comparative genomics of rare sequence types. Front Microbiol. 2016; 7: 270. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYan CZY, Austin CM, Ayub Q, et al.: Genomic characterization of Vibrio parahaemolyticus from Pacific white shrimp and rearing water in Malaysia reveals novel sequence types and structural variation in genomic regions containing the Photorhabdus insect-related (Pir) toxin-like genes. FEMS Microbiol Lett. 2019; 366(17): pii: fnz211. PubMed Abstract \| Publisher Full Text\n\nTheethakaew C, Nakamura S, Motooka D, et al.: Plasmid dynamics in Vibrio parahaemolyticus strains related to shrimp Acute Hepatopancreatic Necrosis Syndrome (AHPNS). Infect Genet Evol. 2017; 51: 211–8. PubMed Abstract \| Publisher Full Text\n\nYang YT, Chen IT, Lee CT, et al.: Draft genome sequences of four strains of Vibrio parahaemolyticus, three of which cause early mortality syndrome/acute hepatopancreatic necrosis disease in shrimp in China and Thailand. Genome Announc. 2014; 2(5): pii: e00816-14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWatson M, Warr A: Errors in long-read assemblies can critically affect protein prediction. Nat Biotechnol. 2019; 37(2): 124–6. PubMed Abstract \| Publisher Full Text\n\nGan HM, Lee YP, Austin CM: Nanopore Long-Read Guided Complete Genome Assembly of Hydrogenophaga intermedia, and Genomic Insights into 4-Aminobenzenesulfonate, p-Aminobenzoic Acid and Hydrogen Metabolism in the Genus Hydrogenophaga. Front Microbiol. 2017; 8: 1880. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAustin CM, Tan MH, Harrisson KA, et al.: De novo genome assembly and annotation of Australia's largest freshwater fish, the Murray cod (Maccullochella peelii), from Illumina and Nanopore sequencing read. GigaScience. 2017; 6(8): 1–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWick RR, Judd LM, Gorrie CL, et al.: Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol. 2017; 13(6): e1005595. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKolmogorov M, Yuan J, Lin Y, et al.: Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol. 2019; 37(5): 540–546. PubMed Abstract \| Publisher Full Text\n\nWalker BJ, Abeel T, Shea T, et al.: Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One. 2014; 9(11): e112963. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWaterhouse RM, Seppey M, Simão FA, et al.: BUSCO Applications from Quality Assessments to Gene Prediction and Phylogenomics. Mol Biol Evol. 2017; 35(3): 543–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHyatt D, Chen GL, Locascio PF, et al.: Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010; 11(1): 119. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTatusova T, DiCuccio M, Badretdin A, et al.: NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 2016; 44(14): 6614–24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlikhan NF, Petty NK, Zakour NLB, et al.: BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics. 2011; 12(1): 402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKelley LA, Mezulis S, Yates CM, et al.: The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015; 10(6): 845–58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUniProt Consortium: UniProt: a hub for protein information. Nucleic Acids Res. 2015; 43(Database issue): D204–D12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFu L, Niu B, Zhu Z, et al.: CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics. 2012; 28(23): 3150–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKatoh K, Standley DM: MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013; 30(4): 772–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrice MN, Dehal PS, Arkin AP: FastTree 2--approximately maximum-likelihood trees for large alignments. PLoS One. 2010; 5(3): e9490. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMuhire BM, Varsani A, Martin DP: SDT: a virus classification tool based on pairwise sequence alignment and identity calculation. PLoS One. 2014; 9(9): e108277. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGan HM, Gan HY, Ahmad NH, et al.: Whole genome sequencing and analysis reveal insights into the genetic structure, diversity and evolutionary relatedness of luxI and luxR homologs in bacteria belonging to the Sphingomonadaceae family. Front Cell Infect Microbiol. 2015; 4: 188. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrehenwinkel H, Wolf M, Lim JY, et al.: Estimating and mitigating amplification bias in qualitative and quantitative arthropod metabarcoding. Sci Rep. 2017; 7(1): 17668. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGan HM, Grandjean F, Jenkins TL, et al.: Absence of evidence is not evidence of absence: Nanopore sequencing and complete assembly of the European lobster (Homarus gammarus) mitogenome uncovers the missing nad2 and a new major gene cluster duplication. BMC Genomics. 2019; 20(1): 335. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGan HM, Linton SM, Austin CM: Two reads to rule them all: Nanopore long read-guided assembly of the iconic Christmas Island red crab, Gecarcoidea natalis (Pocock, 1888), mitochondrial genome and the challenges of AT-rich mitogenomes. Mar Genomics. 2019; 45: 64–71. PubMed Abstract \| Publisher Full Text\n\nXiao J, Liu L, Ke Y, et al.: Shrimp AHPND-causing plasmids encoding the PirAB toxins as mediated by pirAB-Tn903 are prevalent in various Vibrio species. Sci Rep. 2017; 7: 42177. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMahillon J, Chandler M: Insertion sequences. Microbiol Mol Biol Rev. 1998; 62(3): 725–74. PubMed Abstract \| Free Full Text\n\nSkipper KA, Andersen PR, Sharma N, et al.: DNA transposon-based gene vehicles - scenes from an evolutionary drive. J Biomed Sci. 2013; 20(1): 92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSheets JJ, Hey TD, Fencil KJ, et al.: Insecticidal toxin complex proteins from Xenorhabdus nematophilus: structure and pore formation. J Biol Chem. 2011; 286(26): 22742–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPhiwsaiya K, Charoensapsri W, Taengphu S, et al.: A Natural Vibrio parahaemolyticus ΔpirAVp pirBVp+ Mutant Kills Shrimp but Produces neither Pir Vp Toxins nor Acute Hepatopancreatic Necrosis Disease Lesions. Appl Environ Microbiol. 2017; 83(16): pii: e00680-17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliams SL, Jensen RV, Kuhn DD, et al.: Analyzing the metabolic capabilities of a Vibrio parahaemolyticus strain that causes early mortality syndrome in shrimp. Aquaculture. 2017; 476: 44–8. Publisher Full Text\n\nAllnutt T, Yan CZY, Crowley TM, et al.: Commentary: Genome Sequence of Vibrio parahaemolyticus VP152 Strain Isolated From Penaeus indicus in Malaysia. Front Microbiol. 2018; 9: 865. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGilson L, Kuo A, Dunlap PV: AinS and a new family of autoinducer synthesis proteins. J Bacteriol. 1995; 177(23): 6946–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVinoj G, Vaseeharan B, Thomas S, et al.: Quorum-Quenching Activity of the AHL-Lactonase from Bacillus licheniformis DAHB1 Inhibits Vibrio Biofilm Formation In Vitro and Reduces Shrimp Intestinal Colonisation and Mortality. Mar Biotechnol (NY). 2014; 16(6): 707–15. PubMed Abstract \| Publisher Full Text\n\nGan HM, Dailey LK, Halliday N, et al.: Genome sequencing-assisted identification and the first functional validation of N-acyl-homoserine-lactone synthases from the Sphingomonadaceae family. PeerJ. 2016; 4: e2332. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGan HM, Buckley L, Szegedi E, et al.: Identification of an rsh Gene from a Novosphingobium sp. Necessary for Quorum-Sensing Signal Accumulation. J Bacteriol. 2009; 191(8): 2551–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLowe N, Gan HM, Chakravartty V, et al.: Quorum-sensing signal production by Agrobacterium vitis strains and their tumor-inducing and tartrate-catabolic plasmids. FEMS Microbiol Lett. 2009; 296(1): 102–9. PubMed Abstract \| Publisher Full Text\n\nGan HM, Austin C: Nanopore long reads enable the first complete genome assembly of a Malaysian Vibrio parahaemolyticus isolate bearing the pVa plasmid associated with acute hepatopancreatic necrosis disease [Data set]. Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3568485" }	[ { "id": "59964", "date": "17 Feb 2020", "name": "Ballamoole Krishna Kumar", "expertise": [ "Reviewer Expertise I am a microbiologist working on vibrio pathogenesis." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nV. para is not a member of Enterobacteriaceae, that needs correction. It is a Gram‐negative, halophilic bacterium belonging to the family of Vibrionaceae. Basically, this pathogen is the leading cause of seafood‐borne gastroenteritis due to the consumption of raw seafood, not the true pathogen of cultured shrimps. However, this bacterium has recently gained importance following the identification of certain strains of Vibrio parahaemolyticus as the causative agent that caused large scale losses in farmed shrimp production in several parts of the world.\nI am not clear about the true source of MVP1 used as no clear description is provided. Is it isolated from the surface of the infected animal or from haemolymph or hepatopancreas or from soil and water of the shrimp pond that was tested positive for AHPND?\nWhat is the evidence that exists to show that authors have used unfragmented gDNA for library preparation?\nThe genome of Vibrio parahaemolyticus MVP1 is how much comparable to the Reference strain?\nAdditional insecticidal toxin gene generated in the chromosome of MVP1 - does it present in the strains used for the comparative analysis? Is it part of any VpaI identified previously? Do you think just mere presence of such genes would precipitate in the disease/pathogenesis? Could the presence of this gene be linked to demonstrable traits among the isolates?\n\nWhat was the rationale behind the analysis of LuxM/OpaM/AinS family autoinducer?\nThe reference list is biased to the more recent articles published by members of the research group and few older references of others. An up to date literature review is clearly needed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "60806", "date": "10 Mar 2020", "name": "Tomoo Sawabe", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAHPND is one of the important threats for the shrimp aquaculture industry to be prevented worldwide. The authors succeeded to have the complete genome of a Vibrio parahaemolyticus strain MVP1 isolated from a Malaysian aquaculture farm. The study is so important but studies on AHPND should be moved to studies on population dynamics based on complete genomes.\nNanopore and Illumina hybrid assemblies are available commercially. The cost-effective methodology can achieve dozens of strains to be sequenced completely. The current manuscript is likely to trace such published known reports. In this aspect, the manuscript is unlikely to be technically sound.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-2108
https://f1000research.com/articles/7-1848/v1	26 Nov 18	{ "type": "Research Article", "title": "Continuous positive airway pressure in patients with rapid eye movement (REM)-specific obstructive sleep apnea, a retrospective match-controlled chart review", "authors": [ "Rodolfo Soca", "Erica Buchner", "Hrayr Attarian", "Erica Buchner", "Hrayr Attarian" ], "abstract": "Background: Rapid eye movement (REM) obstructive sleep apnea (OSA) represents 13 to 35% of all OSA cases and is more common in women. Continuous positive airway pressure (CPAP) is the gold standard for treatment of all forms of OSA but we do not know if patients with REM OSA have different pressure requirements than those with non-stage dependent OSA. Methods: This was a retrospective case control study. We first identified individuals with REM OSA and then tried to identify apnea hypopnea index (AHI), gender, and body mass index (BMI) matching controls that had non-stage specific OSA. Individuals were considered to have REM OSA if the REM AHI was greater than 5 events/hour, and the ratio of REM AHI / non-rapid eye movement (NREM) AHI was greater than 2. Demographic variables and the recommended CPAP pressure were analyzed using paired Student’s T-Tests. Results: Our study included a total of 16 individuals with REM OSA and equal number of AHI, gender, and BMI matching controls. Both groups had similar demographic and polysomnographic characteristics. Individuals with REM OSA required similar CPAP pressures as controls (7.5 cm H2O vs 7.4 cm H2O p=0.78). Conclusion: Individuals with REM require similar CPAP pressures as their AHI, gender, and BMI matching controls.", "keywords": [ "REM OSA", "CPAP", "obstructive sleep apnea" ], "content": "Introduction\n\nThe prevalence of obstructive sleep apnea (OSA) in adults has been estimated to be between 1% to 2% in women and 3% to 4% in men1. OSA is associated with increased cardiovascular mortality and it is considered an independent risk factor for all cause mortality2–4. The severity of the disease is usually expressed using the apnea hypopnea index (AHI)5.\n\nIn a subset of those with OSA, respiratory events happen predominantly or exclusively during rapid eye movement (REM) sleep; this condition is referred to as “REM-related OSA (REM OSA)”. Its prevalence among patients with OSA is 13 to 35% depending on the criteria used to define AHIs6–8. REM OSA has been a topic of debate in the sleep medicine community since we do not know if this form of sleep apnea is part of the spectrum of “general” sleep apnea or a completely different entity with unique risk factors and different treatment needs.\n\nREM OSA does not have a male predilection, in fact it may be even more common in women. Unlike OSA the likelihood of REM OSA decreases with age7,8. REM OSA is not associated with daytime sleepiness or diminished quality of life (QOL)9.\n\nIt is, however, linked to increased incidence of hypertension10 and type 2 diabetes11.\n\nCPAP is the gold standard for treatment of both REM and NREM OSA, and both forms of sleep apnea seem to respond in similar ways to CPAP therapy.12. We still don’t know if CPAP pressures required to control REM OSA are significantly different than those needed for OSA in general.\n\nWe designed this study with the initial hypothesis that individuals with REM OSA would require a lower CPAP pressure than controls. This hypothesis was based on day-to-day observations at our sleep center.\n\n\nMethods\n\nThe Institutional Review Board (IRB) at HealthEast care system approved this study (IRB #HE1511002). The search included all polysomnography (PSG) tests that were completed from January 1st 2014 to December 31st 2014. We searched all patients older than 18 years old who had a baseline evaluation as well as a CPAP titration. REM OSA subjects and controls were required to have NREM and REM sleep during the baseline test and a minimum of 10 minutes of stage REM sleep during the titration.\n\n\nIdentification of individuals and controls\n\nWe first identified individuals that met criteria for REM OSA using the sleep center’s database. Data was extracted by reviewing the database manually, record by record, and reviewing the polysomnography report; all the variables that were needed for this study were already part of the reports. The dataset that is provided, was used to collect the information on an MS Excel 2013 spreadsheet (Dataset 113). No information regarding CPAP pressure recommendations was accessed during this phase to avoid selection bias. The database was searched a second time in order to identify gender, BMI, and AHI matching controls. Individuals were considered a matching control if the BMI and the AHI were within 5 kg/m2 and 5 events/hour respectively.\n\n\nSample Size and controls\n\nBased on preliminary sample-size calculations, we aimed to identify 50 individuals with REM OSA and 50 controls in order to detect a pressure difference of 1 cwp with a significance level of 0.05 Power 0.8. This was based on a separate estimate of the mean CPAP pressure recommended at the lab (9 cwp; SD=2.45; D=0.41). We only found 16 matching controls.\n\n\nPolysomnography (PSG) values and definitions\n\nPSG tests were scored by a certified PSG technologist the night of the test using the American Academy of Sleep Medicine Scoring Manual v 2.05.\n\n\nREM OSA definition\n\nIndividuals were considered to have REM OSA if the REM AHI was greater than 5 events/hour, and the ratio of REM AHI / non-rapid eye movement (NREM) AHI was greater than 2. REM sleep duration had to be greater than 15 minutes.\n\n\nOSA definition for controls\n\nIndividuals were considered appropriate controls if they had an AHI greater than 5 events/hour and within 5 events/hour of the REM OSA match. The NREM AHI had to be greater than 5 events/hour and the ratio of REM AHI to NREM AHI had to be 1 or less. REM sleep duration had to be greater than 15 minutes.\n\n\nCPAP pressure\n\nThe recommended CPAP pressure was the lowest pressure that eliminated respiratory events during the test, including supine REM sleep.\n\n\nOther variables\n\nWe collected demographic, clinical, and polysomnographical variables such as age, gender, Body mass index (BMI), presence of diabetes, presence of hypertension, recording time, total sleep time, Epworth score (to assess daytime sleepiness), STOP BANG Score (to assess risk for OSA), and percentage of each sleep stage.\n\n\nStatistical analysis\n\nDescriptive characteristics for demographic information were summarized as mean with standard deviation for continuous variables and as frequency for categorical variables.\n\nContinuous and categorical variables were compared using paired Student’s T-Tests and Chi-square tests, respectively. All hypothesis tests were 2-sided, with a significance level of 0.05. All analyses were performed using R® for Mac OS X GUI version 1.67.\n\n\nResults\n\nWe were able to identify a total of 16 individuals with REM OSA who had gender, AHI, and BMI matching controls for a total of 32 subjects in the study. Table 1 summarizes the demographic and clinical characteristics of individuals with REM OSA and controls.\n\na- ESS= Epworth Sleepiness Scale Score\n\nb- HI= Hypopnea Index\n\nc- CAI= Central Apnea Index\n\nd- REM OSA = Rapid eye movement related obstructive sleep apnea\n\ne- NREM = Non rapid eye movement\n\nf- AHI = Apnea hypopnea index\n\ng- ODI = Oxygen desaturation index\n\nBoth groups were very similar in terms of age, daytime sleepiness, diabetes, hypertension, and sleep architecture parameters, . Subjects with REM-OSA had a slightly lower score in the STOP-BANG questionnaire (3.5 vs. 4.0 p= 0.03).\n\nFigure 1 shows the recommended CPAP pressure for the REM OSA group and controls. There was no significant difference between the groups.\n\n\nDiscussion\n\nTo our knowledge our study is the first comparing nPAP treatment requirements for individuals with REM OSA. We found that individuals with REM OSA required similar nCPAP pressures as gender, AHI, and BMI matched controls.\n\nThis was a very simple and limited study that was designed to test our hypothesis that individuals with REM OSA required lower CPAP pressures than controls. There has been debate about whether REM OSA constitutes a different phenomenon or is just very similar to any other form of OSA. Our results suggest that, REM OSA is not different than NREM OSA.\n\nAge, gender, and AHI have been identified as predictors of CPAP pressure needs but none of those studies have looked at the stage in which the respiratory events happen.14,15. Since REM OSA has a different gender and BMI association than the general OSA population, we were able to isolate the effect of the REM component by looking at gender, AHI, and BMI matched controls16,17.\n\nOur study had the limitation of a small sample size because it was difficult to identify an adequate number of matching controls. With our small sample size, the study was underpowered to detect smaller differences in CPAP pressure needs. We still think that publishing our results could be important because there are no other studies, regardless of sample size, that have looked into this topic.\n\n\nData availability\n\nF1000Research: Dataset 1. Rapid eye movement related obstructive sleep apnea (REM OSA) dataset, https://doi.org/10.5256/f1000research.16688.d22633113", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nYoung T, Palta M, Dempsey J, et al.: The occurrence of sleep-disordered breathing among middle-aged adults. N Engl J Med. 1993; 328(17): 1230–5. PubMed Abstract \| Publisher Full Text\n\nPunjabi NM, Caffo BS, Goodwin JL, et al.: Sleep-disordered breathing and mortality: a prospective cohort study. PLoS Med. 2009; 6(8): e1000132. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarin JM, Carrizo SJ, Vicente E, et al.: Long-term cardiovascular outcomes in men with obstructive sleep apnoea-hypopnoea with or without treatment with continuous positive airway pressure: an observational study. Lancet. 2005; 365(9464): 1046–53. PubMed Abstract\n\nDaviglus ML, Talavera GA, Avilés-Santa ML, et al.: Prevalence of major cardiovascular risk factors and cardiovascular diseases among Hispanic/Latino individuals of diverse backgrounds in the United States. JAMA. 2012; 308(17): 1775–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMedicine AAoS: The AASM Manual for the Scoring of Sleep and Associated Events. v 2.2 ed. 2015. Reference Source\n\nHaba-Rubio J, Janssens JP, Rochat T, et al.: Rapid eye movement-related disordered breathing: clinical and polysomnographic features. Chest. 2005; 128(5): 3350–7. PubMed Abstract \| Publisher Full Text\n\nKoo BB, Dostal J, Ioachimescu O, et al.: The effects of gender and age on REM-related sleep-disordered breathing. Sleep Breath. 2008; 12(3): 259–64. PubMed Abstract \| Publisher Full Text\n\nKoo BB, Patel SR, Strohl K, et al.: Rapid eye movement-related sleep-disordered breathing: influence of age and gender. Chest. 2008; 134(6): 1156–61. PubMed Abstract \| Publisher Full Text\n\nChami HA, Baldwin CM, Silverman A, et al.: Sleepiness, quality of life, and sleep maintenance in REM versus non-REM sleep-disordered breathing. Am J Respir Crit Care Med. 2010; 181(9): 997–1002. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMokhlesi B, Finn LA, Hagen EW, et al.: Obstructive sleep apnea during REM sleep and hypertension. results of the Wisconsin Sleep Cohort. Am J Respir Crit Care Med. 2014; 190(10): 1158–67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrimaldi D, Beccuti G, Touma C, et al.: Association of obstructive sleep apnea in rapid eye movement sleep with reduced glycemic control in type 2 diabetes: therapeutic implications. Diabetes Care. 2014; 37(2): 355–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSu CS, Liu KT, Panjapornpon K, et al.: Functional outcomes in patients with REM-related obstructive sleep apnea treated with positive airway pressure therapy. J Clin Sleep Med. 2012; 8(3): 243–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSoca R, Buchner E, Attarian H: Dataset 1 in: Continuous positive airway pressure in patients with rapid eye movement (REM)-specific obstructive sleep apnea, a retrospective match-controlled chart review. F1000Research. 2018. http://www.doi.org/10.5256/f1000research.16688.d226331\n\nMiljeteig H, Hoffstein V: Determinants of continuous positive airway pressure level for treatment of obstructive sleep apnea. Am Rev Respir Dis. 1993; 147(6 Pt 1): 1526–30. PubMed Abstract \| Publisher Full Text\n\nStradling JR, Hardinge M, Smith DM: A novel, simplified approach to starting nasal CPAP therapy in OSA. Respir Med. 2004; 98(2): 155–8. PubMed Abstract \| Publisher Full Text\n\nYukawa K, Inoue Y, Yagyu H, et al.: Gender differences in the clinical characteristics among Japanese patients with obstructive sleep apnea syndrome. Chest. 2009; 135(2): 337–343. PubMed Abstract \| Publisher Full Text\n\nJayaraman G, Majid H, Surani S, et al.: Influence of gender on continuous positive airway pressure requirements in patients with obstructive sleep apnea syndrome. Sleep Breath. 2011; 15(4): 781–4. PubMed Abstract \| Publisher Full Text" }	[ { "id": "49943", "date": "03 Jul 2019", "name": "Ludovico Messineo", "expertise": [ "Reviewer Expertise Obstructive sleep apnea" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this simple study, Roca and colleagues compare CPAP requirements in patients with obstructive sleep apnea (OSA) and rapid eye movement (REM) related OSA. Their conclusion is that both groups require similar therapeutic CPAP levels.\nAlthough the authors acknowledge that their study is underpowered and thus potentially unable to detect any statistical difference between the two groups (also considering the retrospective design), the main message of the manuscript results are too strong given such preliminary results. Therefore, I would suggest some adjusting in wording to put less emphasis on the clinical outcome of this work (i.e. in the abstract: “Individuals with REM might require similar CPAP pressures”; in the discussion: “Our results suggest that, REM OSA could be not different than NREM OSA for what concern CPAP needs”).\nFurthermore, I have a number of other concerns:\nSu and colleagues found lower CPAP requirements following titration in a bigger, non-AHI matched population of REM-OSA vs OSA,1 which would deserve a comment in the discussion. The very high REM AHI in the REM OSA group (was this high number driven by people with the shortest amount of REM?) could lead to an out-of-proportion increase of CPAP at the titration (especially if this was done automatically, see also next point) and consequently to higher average CPAP requirements for the whole group. Consider discuss this further as a potential bias for the results. How were the recommended CPAP levels obtained? After titration (and, if so, automatic CPAP or manual titration) or after follow-up? For OSA prevalence, much more recent estimates exist. A quote is missing at the end of the second paragraph. Define acronyms before using them (i.e. cwp).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5082", "date": "13 Dec 2019", "name": "rodolfo soca", "role": "Author Response", "response": "We thank Dr. Ludovico Messineo for taking the time to review our article and providing valuable feedback about our paper.We have rewritten our manuscript to incorporate the main points from Dr. Messineo’s review as explained below: The main suggestion from Dr. Messineo relates to the strong message that was conveyed despite the fact that our study had a small sample size. We agree with the suggestion completely. We changed the wording in the abstract to covey the fact that our study “suggests” that CPAP pressure needs could be similar between REM and NREM OSA. We also changed the wording of our discussion section to convey the same message. We agree with the suggestion about including more information about the lower CPAP pressure needs that were identified by Su et al. We added some wording to the introduction and to the discussion to mention their results and our explanation as to why we think that we obtained different results: their study had a high proportion of women in the REM OSA group. Regarding the relationship between REM duration, REM AHI, and recommended CPAP pressures, we agree with the reviewer that the topic is interesting but we decided to address it in these comments rather than adding more text to our results. While we found a negative correlation between REM sleep duration and REM AHI for the REM OSA group (Pearson’s coefficient r =- 0.56), we did not find a relationship between the final CPAP pressure and the duration of REM sleep (Pearson’s coefficient r = -0.04). We thank the reviewer for noting our limited explanation regarding the recommended CPAP pressure. We added wording to the methods section to explain that the recommended CPAP pressure was obtained manually during the CPAP titration study. We added some wording to highlight the fact that our prevalence data comes from the Wisconsin Sleep Cohort study. We thank the reviewer for noting that a quote was missing at the end of our second paragraph, this was certainly a very relevant reference for us. Reference added. We apologize for the use of an acronym that was not defined. We updated our manuscript and changed “cwp” with the appropriate and more standard unit “cm H2O” Once again, we thank the reviewer for this insightful review and welcome any comments about this newer version of the manuscript.Very respectfully, The Authors." } ] } ]	1	https://f1000research.com/articles/7-1848
https://f1000research.com/articles/8-1707/v1	01 Oct 19	{ "type": "Research Article", "title": "In silico analysis of a major allergen from Rattus norvegicus, Rat n 1, and cross-reactivity with domestic pets", "authors": [ "Marlon Munera", "Neyder Contreras", "Andres Sánchez", "Jorge Sánchez", "Yuliana Emiliani", "Neyder Contreras", "Andres Sánchez", "Jorge Sánchez", "Yuliana Emiliani" ], "abstract": "Background: Lipocalins play a role in the cellular trafficking of pheromones and are involved in allergic responses to domestic pets. However, the cross-reactivity among allergens of this group has been poorly explored, and the pheromone linking capacity is not well characterized. The aim of this study was to explore cross-reactive epitopes and pheromone linking capacity among Rat n 1 and homologues in domestic pets through an in silico approach. Methods: ElliPro and BepiPred in silico tools were used to predict B cell linear and cross-reactive epitopes. The pheromone linking capacity was explored by docking virtual screening with 2-ethylhexanol, 2,5-dimethylpyrazine, 2-sec-butyl-4,5-dihydrothiazole, and 2-heptanone ligands. Results: According to the analysis, Rat n 1 shares 52% identity with Equ c 1, Can f 6, Fel d 4, and Mus m 1 allergens. The overlapping structures assay revealed high structural homology (root mean square deviation < 1). Four lineal and three discontinuous epitopes were predicted on Ra t n 1. A lineal epitope located between amino acids residues 24 and 36 was highly conserved on all allergens explored. A cross-reactive discontinuous epitope (T142, K143, D144, L145, S146, S147, D148, K152, L170, T171, T173, D174) was also found. Docking molecular simulations revealed the active site, and we identified the properties of the binding of four pheromones and the binding potential of Rat n 1. Critical residues for interactions are reported in this study. Conclusions: We identified some possible allergens from Rattus norvegicus, and those allergens could have cross-reactivity with allergens from some animals. The results need to be confirmed with in vitro studies and could be utilized to contribute to immunotherapy and reduce allergic diseases related to lipocalins.", "keywords": [ "Allergen", "lipocalin", "cross-reactivity", "docking", "in silico" ], "content": "Introduction\n\nLipocalins are among the most important indoor/outdoor groups of animal allergens. For some, the protein structure has been resolved, but their functions are still elusive. Lipocalins generally display a low sequence identity between family members1, but the lipocalin allergens are usually well preserved and can present similar patches that, in addition to serum albumins, may contribute to allergic cross-reactions among furry animals2–4. Rodents, especially mice and rats, are cosmopolitan species present in rural, periurban, and urban areas, and are most often considered as pests. In addition, the presence of these species as pets in homes and their permanent use as animal models in research laboratories have allowed constant exposure to their allergens, which is an important source of sensitization5,6.\n\nSo far, only one rat allergen has been described, Rat n 1, which is a lipocalin formed by two fractions, a prealbumin and an α-2U-globulin, secreted by the liver and found in high concentrations in urine, but also in saliva and fur7,8. Among patients allergic to rats, 73% and 87% reacted to Rat n 1 in dust9. This molecule is glycosylated and, up to now, was known to have two isoforms: Rat n 1.01 (21 kDa) and Rat n 1.02 (17 kDa). Its structure is like a conventional lipocalin with eight antiparallel β chains forming a single beta sheet and an α helix to create a pocket for ligand binding, very similar to that of other allergens, such as Mus m 1, the mouse's main allergen, with a highly conserved identity5,10,11. Four regions with potential immunodominant T cell epitopes have been described, and three of these are co-localized with the conserved regions of lipocalin, similar to the epitopes found in Bos d 2. No B cell epitopes have been reported for Rat n 112.\n\nAlthough its structure and sensitization capacity have been well described, little is known about its biological functions and how these may be related to hypersensitivity mediated by an IgE measured response and cross-reactivity with the main allergens of the most common domestic animals, dogs, cats, and horses13,14. Therefore, the objective of this study was to explore cross-reactive epitopes among Rat n 1 and homologues in domestic pets through an in silico approach.\n\n\nMethods\n\nThe amino acid sequences of lipocalins from 5 domestic animals (Rat n 1, Mus m 1, Fel d 4, Can f 6, and Equ c 1) were selected based on the reported allergenic and phylogenetic capacity15. The sequences were obtained from the UniProt database (Table 1). Sequences that were reported by the World Health Organization (WHO)/International Union of Immunological Societies (IUIS) Allergen Nomenclature Subcommittee and had complete sequences were used. Identity grades among lipocalins used in this study were determined by using the PRALINE web server16. Parameters to perform alignment were set up to use BLOSUM62 as an exchange matrix. Three iterations were used, with an E-value of 0.01. Structural homology and root mean square deviation values were determined using UCSF Chimera (V. 1.13.1) and PDB Viewer software (v.4.10)17.\n\nA model of the Fel d 4 allergen was made by homology using the SWISS-MODEL server. The quality of the model was analyzed by ProSA-web. The model was refined in DeepView v.4.1 (energy minimization and rotamer replacements). Its quality was evaluated by several tools, including Ramachandran graphs, WHATIF, QMEAN4 index, and energy values (GROMOS96 force field). Three-dimensional structures of Rat n 1, Mus m 1, Can f 6, and Equ c 1 were retrieved from the Protein Data Bank.\n\nElliPro and BepiPred tools were used to predict discontinuities and lineal epitopes on Rat n 118. With ElliPro, the 3D structure of Rat n 1 was used to predict epitopes. Minimum score and maximum distance (Angstrom) were set to 0.5 and 6.\n\nPreparation of receptors and ligands was carried out using the freely available Discovery Studio Visualizer 2016. Treatment of the receptors consisted of extracting the ligand and eliminating water molecules and cofactors with which their crystalline structures are resolved, followed by preparation of the ligands, making corrections in the structures, generating variations, and eliminating unwanted structures. Adding hydrogen atoms, neutralizing charged groups, generating ionization and tautomer states, obtaining alternative chiralities, and optimizing geometries were carried out.\n\nUsing molecules identified as pheromones and the 3-dimensional molecular modeling of odorant binding protein (OBP1), docking studies were performed using SwissDock based on EADock DSS, in the following stages: (1) generation of binding modes in local and blind docking, (2) estimation of CHARMM force field energies with GRID, (3) binding of modes with the most favorable energies with FACTS and clusters, and (4) visualization of the most favorable clusters. The best-scoring docked models exhibiting the best superposition with ligands and lowest binding energy were analyzed and visualized with Chimera (V.1.13.1).\n\nThe Rat n 1 3D structure was submitted to the ConSurf server in order to generate evolutionarily related conservation scores to help to identify functional regions in the proteins. Functional and structural critical residues in Rat n 1 sequence were confirmed by the ConSeq server.\n\n\nResults\n\nMultiple alignment among amino acid sequences from Rat n 1, Can f 6, Equ c 1, Fel d 4, and Mus m 1 was performed. A 62% identity was identified among sequences compared. Residues located on positions 29 to 73 showed the highest identity. The sequence alignments of the lipocalins showed that identical residues formed short continuous segments (Figure 1). A comparison of the secondary structural elements of Rat n 1 with the structures listed in Table 1 revealed backbone atomic RMSD values between 0.3 and 0.95 Å, with Mus m 1 showing the most closely related structure and sequence homology to Rat n 1. For all structures analyzed, the closest structural homology was found on the α-helical amino acid sequence spanning region on Rat n 1 containing nine conserved residues (IKEKFAK-L) (Figure 2). While these proteins showed the same overall fold change, some detailed structures contained differences, such as major structural differences located on loop regions for all allergens in this study.\n\nLipocalins share a 62% of identity in their amino acid sequences.\n\n(A) Rat n 1 (Blue) and Mus m 1 (Brown). (B) Rat n 1 and Fel d 4 (Green). (C) Rat n 1 and Equ c 1 (Violet). (D) Rat n 1 and Can f 6 (Orange). RMSD values are showed in Å.\n\nUsing ElliPro and BepiPred servers, four lineal and three discontinuous epitopes on Rat n 1 were predicted (Table 2 and Table 3). The first and third epitopes were located on α-helices, spanning residues 158–165 and 141–148 (Figure 3). Both epitopes had a surface area of 300 Å.\n\nLE (Lineal epitope).\n\nLE (Discontinous Epitope).\n\n(A–C) Surface models showing areas covered by predicted epitopes 1-4. (D–F) Cartoon models showing location of predicted epitopes on 3D structure.\n\nThe third epitope was identified as being cross-reactive among Rat n 1, Mus m 1, Equ c 1, Can f 6, and Fel d 4. From all residues conforming with mapped epitopes, 80% were conserved and surface exposed among lipocalins analyzed in this study. The second and fourth epitopes were located on loop regions, spanning residues 91–97 and 24–36 with surface areas of 262 and 487 Å, respectively (Figure 3). Conservative analysis indicated that both regions were highly conserved in the lipocalin family (Figure 4). According to ConSurf analysis, the region covering the second lineal epitope is conserved among the lipocalin family.\n\n(A–C) Ribbon models showing conserved region among lipocalins. (D–F) Surface models showing conserved region among lipocalins. (G–I) Cartoon models showing linear epitopes predicted.\n\nThe first and second discontinuous epitopes were constituted 10 amino acid residues with a surface area of 375 Å; the first discontinuous epitope was distributed on G-H and F-E β-strands and loops connecting them,where as the third epitope was mapped to an α-helical, the same region where the first lineal epitope was located (Figure 5). This epitope contained 12 amino acid residues, and of these, 85% were surface exposed with a surface area of 487 Å.\n\n(A, B) Surface models showing discontinuous epitopes predicted by Ellipro server. (C–E) Cartoon models showing location of residues conforming epitopes discontinues.\n\nDocking molecular simulations were conducted to reveal the active sites, identify the binding properties of four pheromones and the binding potential of Rat n 1. In the docked complexes (Figure 6 and Figure 7), the central region of the corresponding structures indicated a step involving cleavage of the protein with aromatic amino acids, specifically Tyr139, Tyr103, Phe73, Phe75, and Phe122. This docked position revealed that the aliphatic structures, pyrazine derivatives, and 2-sec-butyl-4,5-dihydrothiazole (SBT) had the lowest bond energies, and the least in 2-heptanone and 2-ethylhexanol (Table 4). Likewise, in 2,5-dimethylpyrazine the interactions of aromatic and aliphatic residues such as Met61, Leu88, Val101, Val137, and Tyr139 are described, which maintain binding to the pyrazine ring and methyl substituents in C2 and C5 (Figure 6A–B). In the case of pheromones such as 2-ethylhexanol, a structural arrangement at the site was shown to be in contact with the aromatic and hydroxyl groups in the structure, which were shown in residues as Phe73, Phe109, Phe122 and Tyr139, not greater than 3.3 Å in angular distance (Figure 6C). The SBT structure was in a specific orientation with the thiazoline ring in the proximal opening of the active site. Likewise, the presence of hydrophobic interactions with apolar and aromatic residues with SBT has been established, in which alkyl-type bonds and π-alkyl are described in Met57, Val59, Leu88, and Leu124 with the thiazoline ring and structural side chain (see Figure 6D). In general, the rest of the carbon structure and the radical presentation of a structural orientation in the anterior site of the pocket in the opposite direction to the β-barrel demonstrated a relationship with the apolar residues between ethyl radicals and interactions type alkyl with Leu124, Leu135, and Val137. Similarly, 2-heptanone showed closer interaction with the protein cavity, predominantly by apolar and polar residues through hydrogen bonds, where it is common to see the relationship between the oxygen of the carbonyl group and aromatic residues of Phe75, Tyr103, and Val101; however, hydrophobic type alkylic junctions were shown with residues such as Leu124 and Val137 (Figure 7).\n\nA detailed view showed the docked complex with the key amino acids involved in the active site of Rat n 1 with pheromones. (A) Docked complex of Rat n 1 – 2,5-Dimethylpyrazine (DMP). (B) Docked complex of Rat n 1 – 2-Ethylhexanol. (C) Docked complex of Rat n 1 – 2-Heptanone. (D) Docked complex of Rat n 1 – 2-sec-butyl-4,5-dihydrothiazole (SBT).\n\n\nDiscussion\n\nAnimal allergens remain an important cause of sensitization and allergic diseases. Rodents such as rats are invasive cosmopolitan species that move between urban, periurban, and urban areas looking for favorable habitats and resources. The allergenicity of these species was first observed in animal caretakers and was considered an important source of occupational sensitization, affecting up to 15% of people in European countries with an active scientific community12. Besides exposure in occupational settings, rodent exposure also occurs in domestic environments, as was shown in inner-city children with asthma in the USA, where rat sensitization rates were 19–21%19. In contrast, a recent study from Europe reported a very low prevalence of sensitization to rodents of 0.59% in urban atopic populations without occupational exposure5. Rat n 1 is the largest allergen of this species and has been well characterized. This protein belongs to the lipocalin family, a transport protein of hydrophobic ligands as lipid signaling molecules.\n\nSeveral major allergens are members of the lipocal infamily, including those from the mouse (Mus m 1), dog (Can f 1 and Can f 2), cat (Fel d 4 and Fel d 7), horse (Equ c 1), cow (Bos d 2 and Bos d 5), hamster (Pho 21kD), and rabbit (Ory c 1 and Ory c 4), among others3. The factors that give rise to so many lipocalins becoming inducers of allergy are unclear.Among the allergens, cross-reactivity has been observed, mainly in organisms to which people are most exposed, such as cats, dogs, and horses.\n\nIn this study, we observed by in silico analysis possible cross-reactivity between Rat n 1, Can f 6, Equ c 1, and Fel d 4, with 62% identity among sequences compared. In previous studies, we found a high conservation state among Rat n 1, Equ c 1, and Fel d 4 (60% identity) and described possible residues with antigenic potential15,20. Nilson et al.21 found cross-reactivity between Can f 6, Fel d 4, and Equ c 1 by inhibition assays, especially in residues located on positions 29 to 73, which showed the highest identity. In these positions, Rat n 1 also presented the highest identity with the other three lipocalins, and here we found a possible lineal epitope (LE4: Start 24 – End 36) that could explain the cross-reactivity. Jeal et al.12 studied a population of individuals exposed to laboratory rats to determinate the proliferative response of peripheral blood mononuclear cells to Rat n 1. They found four regions as possible immunodominant T cell epitopes, and three of them were localized within the conserved regions of the lipocalins. One was also found in our study as a possible lineal epitope (LE2: Start 91 – End 97), with a high identity with Mus m 1, Equ c 1, Can f 6, and Fel d 4. The homologous allergens may contribute to multisensitization and symptoms in individuals allergic to different animals. Also, cross-reactivity to T cell epitope was found between Can f 1 and human tear lipocalin22. This could support the autosensitization and increased inflammatory response mediated by T lymphocyte CD4+.\n\nThe docking simulations demonstrated that the Rat n 1 cleft is big enough to accommodate the whole fatty acid molecule. Determining the capacity to bind some ligands by allergens is critical to understand their allergenic capacity. For Bet v 1, a lipocalin family member, it has been determined that ligands such as lipids, iron, and calcium modulate its allergenicity capacity23,24. When Bet v 1 is properly loaded with iron, it can promote Th2 response. A similar property is reported for Pru p 3, a peach lipid transfer protein. Results reveal that the ligand is recognized by a type of cellular receptor called CD1d in the cell surface where the antigens appear, that is, substances able to provoke an immune system response to produce antibodies. CD1d is responsible for presenting lipid antigen to activating cells of the immune system called invariant natural killer T (iNKT) cells. Once activated, these iNKT cells produce an enormous amount of substances that cause the characteristic symptoms of allergic disorders25. Since many allergens transport diverse compounds, the discovery of Pru p 3 lipid-ligand as an adjuvant to promote allergic sensitization through its recognition by CD1d expression opened new horizons25.\n\nOf the lipocalin family, only Mus m 1 has been characterized for pheromone linking. Experimental assays revealed that 2-sec-butyl-4,5-dihydrothiazole (SBT), 6-hydroxy-6-methyl-3-heptanone (HMH), and (±)-dehydro-exo-brevicomin (DHB) are ligands for Mus m 1. This was a first step in determining ligands with allergenic capacity26. Also, some ligands could influence the stabilization of IgE conformational epitopes23,27. Here, we predicted three. So, experimental assays are needed to determine the impact of ligands in Rat n 1 on inducing allergic responses.This is the first exploration about capacity of Rat n 1 to bound pheromones ligands. For other allergens, such as Fel d 1, lipid ligands enhance TLR4 activation and innate immune signaling and promote airway hypersensitivity reactions in diseases such as asthma28.\n\nThe outcomes of the current work include (1) a comprehensive understanding of the structure of Rat n 1 protein and structural similarities and differences between Rat n 1 and other lipocalins, and (2) a structural and molecular basis for the identification of epitopes responsible for cross-allergenicity between rat and domestic animal allergenic lipocalins. These epitopes may contribute significantly to designing rational strategies for diagnosis of and immunotherapy for domestic animal allergies.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nFlower DR, North AC, Sansom CE, et al.: The lipocalin protein family: structural and sequence overview. Biochim Biophys Acta. 2000; 1482(1–2): 9–24. PubMed Abstract \| Publisher Full Text\n\nKonradsen JR, Fujisawa T, van Hage M, et al.: Allergy to furry animals: New insights, diagnostic approaches, and challenges. J Allergy Clin Immunol. 2015; 135(3): 616–25. PubMed Abstract \| Publisher Full Text\n\nSánchez A, Cardona R, Sánchez J: Análisis in silico de lipocalinas de perro, gato, caballo, vaca, hámster y gallina. Posible efecto en el estudio de las enfermedades alérgicas. Revista Alergia México. 2016; 63(1): 1–10. Publisher Full Text\n\nGergen PJ, Mitchell HE, Calatroni A, et al.: Sensitization and Exposure to Pets: The Effect on Asthma Morbidity in the US Population. J Allergy Clin Immunol Pract. 2018; 6(1): 101–107 e2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiccardi G, Salzillo A, Sofia M, et al.: Sensitization to rodents (mouse/rat) in an urban atopic population without occupational exposure living in Naples, Italy. Eur Ann Allergy Clin Immunol. 2012; 44(5): 200–4. PubMed Abstract\n\nFishbein AB, Lee TA, Cai M, et al.: Sensitization to mouse and cockroach allergens and asthma morbidity in urban minority youth: Genes-environments and Admixture in Latino American (GALA-II) and Study of African-Americans, Asthma, Genes, and Environments (SAGE-II). Ann Allergy Asthma Immunol. 2016; 117(1): 43–49 e1. PubMed Abstract \| Publisher Full Text\n\nBayard C, Holmquist L, Vesterberg OJ, et al.: Purification and identification of allergenic alpha (2u)-globulin species of rat urine. Biochim Biophys Acta. 1996; 1290(2): 129–134. PubMed Abstract \| Publisher Full Text\n\nGordon S, Tee RD, Stuart MC, et al.: Analysis of allergens in rat fur and saliva. Allergy. 2001; 56(6): 563–567. PubMed Abstract \| Publisher Full Text\n\nGordon S, Tee RD, Newman Taylor AJ, et al.: Analysis of the allergenic composition of rat dust. Clin Exp Allergy. 1996; 26(5): 533–541. PubMed Abstract\n\nJeal H, Harris J, Draper A, et al.: Dual sensitization to rat and mouse urinary allergens reflects cross-reactive molecules rather than atopy. Allergy. 2009; 64(6): 855–861. PubMed Abstract \| Publisher Full Text\n\nBjerg A, Winberg A, Berthold M, et al.: A population-based study of animal component sensitization, asthma, and rhinitis in schoolchildren. Pediatr Allergy Immunol. 2015; 26(6): 557–63. PubMed Abstract \| Publisher Full Text\n\nJeal H, Draper A, Harris J, et al.: Determination of the T cell epitopes of the lipocalin allergen, Rat n 1. Clin Exp Allergy. 2004; 34(12): 1919–1925. PubMed Abstract \| Publisher Full Text\n\nRaulf M: Allergen component analysis as a tool in the diagnosis and management of occupational allergy. Mol Immunol. 2018; 100: 21–27. PubMed Abstract \| Publisher Full Text\n\nUriarte SA, Sastre J: Clinical relevance of molecular diagnosis in pet allergy. Allergy. 2016; 71(7): 1066–8. PubMed Abstract \| Publisher Full Text\n\nMarlon M, Andres S, Jorge S, et al.: In Silico Analysis of Cross Reactivity between Lipocalin of Domestic Animals. Open J Immunol. 2018; 08(04): 97–106. Publisher Full Text\n\nSimossis VA, Heringa J: PRALINE: a multiple sequence alignment toolbox that integrates homology-extended and secondary structure information. Nucleic Acids Res. 2005; 33(Web Server issue): W289–94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPettersen EF, Goddard TD, Huang CC, et al.: UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004; 25(13): 1605–12. PubMed Abstract \| Publisher Full Text\n\nPonomarenko J, Bui HH, Li W, et al.: ElliPro: a new structure-based tool for the prediction of antibody epitopes. BMC Bioinformatics. 2008; 9: 514. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPerry T, Matsui E, Merriman B, et al.: The prevalence of rat allergen in inner-city homes and its relationship to sensitization and asthma morbidity. J Allergy Clin Immunol. 2003; 112(2): 346–352. PubMed Abstract \| Publisher Full Text\n\nMúnera M, Sanchez A, Sánchez J, et al.: Allergy to Mus m 1: Allergy to Mus m 1: A review of structural, and immunological features. Immunol Lett. 2019; 209: 1–3. PubMed Abstract \| Publisher Full Text\n\nNilsson OB, Binnmyr J, Zoltowska A, et al.: Characterization of the dog lipocalin allergen Can f 6: the role in cross-reactivity with cat and horse. Allergy. 2012; 67(6): 751–757. PubMed Abstract \| Publisher Full Text\n\nLiukko ALK, Kinnunen TT, Rytkönen-Nissinen MA, et al.: Human CD4+ T cell responses to the dog major allergen Can f 1 and its human homologue tear lipocalin resemble each other. PLoS One. 2014; 9(5): e98461. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAsam C, Batista AL, Moraes AH, et al.: Bet v 1--a Trojan horse for small ligands boosting allergic sensitization? Clin Exp Allergy. 2014; 44(8): 1083–93. PubMed Abstract \| Publisher Full Text\n\nRoth-Walter F, Gomez-Casado C, Pacios LF, et al.: Bet v 1 from birch pollen is a lipocalin-like protein acting as allergen only when devoid of iron by promoting Th2 lymphocytes. J Biol Chem. 2014; 289(25): 17416–17421. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScheurer S, Schülke S: Interaction of Non-Specific Lipid-Transfer Proteins With Plant-Derived Lipids and Its Impact on Allergic Sensitization. Front Immunol. 2018; 9: 1389: PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSharrow SD, Vaughn JL, Zídek L, et al.: Pheromone binding by polymorphic mouse major urinary proteins. Protein Sci. 2002; 11(9): 2247–2256. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJimenez-Lopez JC, Robles-Bolivar P, Lopez-Valverde FJ, et al.: Ole e 13 is the unique food allergen in olive: Structure-functional, substrates docking, and molecular allergenicity comparative analysis. J Mol Graph Model. 2016; 66: 26–40. PubMed Abstract \| Publisher Full Text\n\nHerre J, Grönlund H, Brooks H, et al.: Allergens as immunomodulatory proteins: the cat dander protein Fel d 1 enhances TLR activation by lipid ligands. J Immunol. 2013; 191(4): 1529–35. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "54531", "date": "14 Oct 2019", "name": "Anna Pomés", "expertise": [ "Reviewer Expertise Allergen characterization and epitope mapping." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Munera et al. performed an in silico analysis of the major rat allergen Rat n 1, and explored cross-reactive epitopes and pheromone linking capacity among Rat n 1 and homologous allergens from domestic pets. The analysis is interesting from a molecular-structural point of view, but the paper needs to be put in context in the allergy field, including previous literature.\nThe structures of Rat n 1 and Mus m 1 were published in Nature in 1992 and reported “the role of these proteins in pheromone transport” and elaborated on “the structural basis of ligand binding”. This publication should be cited, and the information about the ligand binding taken into consideration for the analysis performed. For example, how the ligands analyzed compare to the pheromones reported in the paper would be very interesting. This is the reference: Böcskei Z, Groom CR, Flower DR, Wright CE, Phillips SEV, Cavaggioni A, Findlay JBC, North ACT. Pheromone binding to two rodent urinary proteins revealed by X-ray crystallography. Nature 1992; 360:186–188.1 Therefore, the sentence in page 9: “Of the lipocalin family, only Mus m 1 has been characterized for pheromone linking” needs correction.\n\nAnother lipocalin whose ligand was identified is cockroach Bla g 4, and this study should be cited and the ligand analyzed and compared with the ones used in this study for a comment in the discussion. Ref: The major cockroach allergen Bla g 4 binds tyramine and octopamine. Offermann LR, Chan SL, Osinski T, Tan YW, Chew FT, Sivaraman J, Mok YK, Minor W, Chruszcz M. Mol Immunol. 2014 Jul;60(1):86-94.2\n\nMapping of B and T cell epitopes has been performed before for some lipocalins (i.e. Dr. Virtanen’s group in Finland). It would be good if the in silico analysis could be related to experimentally identified epitopes, and to discuss the results in the context of previous studies, such as:\nBayard C, Siddique AB, Berzins K, Troye-Blomberg M, Hellman U, Vesterberg O. Mapping of IgE binding regions in the major rat urinary protein, alpha 2u-globulin, using overlapping peptides. Immunol Invest. 1999 Sep-Dec;28(5-6):323-38.3\nZeiler T, Mäntyjärvi R, Rautiainen J, Rytkönen-Nissinen M, Vilja P, Taivainen A, Kauppinen J, Virtanen T. T cell epitopes of a lipocalin allergen colocalize with the conserved regions of the molecule. J Immunol. 1999 Feb 1;162(3):1415-22.4\nKinnunen T, Buhot C, Närvänen A, Rytkönen-Nissinen M, Saarelainen S, Pouvelle-Moratille S, Rautiainen J, Taivainen A, Maillère B, Mäntyjärvi R, Virtanen T. The immunodominant epitope of lipocalin allergen Bos d 2 is suboptimal for human T cells. Eur J Immunol. 2003 Jun;33(6):1717-26.5\nImmonen A, Farci S, Taivainen A, Partanen J, Pouvelle-Moratille S, Närvänen A, Kinnunen T, Saarelainen S, Rytkönen-Nissinen M, Maillere B, Virtanen T. T cell epitope-containing peptides of the major dog allergen Can f 1 as candidates for allergen immunotherapy. J Immunol. 2005 Sep 15;175(6):3614-20.6\nImmonen A, Kinnunen T, Sirven P, Taivainen A, Houitte D, Peräsaari J, Närvänen A, Saarelainen S, Rytkönen-Nissinen M, Maillere B, Virtanen T. The major horse allergen Equ c 1 contains one immunodominant region of T cell epitopes. Clin Exp Allergy. 2007 Jun;37(6):939-47.7\n\nThe rationale for selecting Can f 6 for the study should be explained since there are other dog lipocalins (Can f 1, Can f 2, Can f 4).\n\nAbstract-results: a) “The overlapping structures assay”. The use of “analysis” instead of “assay” is best.\nb) “docking …revealed the active site”. The expression “active site” is especially used for enzymes, and for binding without catalytic activity involved the term “binding site” might be more appropriate. The authors need to correct where it applies (e.g. page 5, line 21).\nc) If Rat n 1 shares 52% amino acid identity with each of the four allergens, then the sentence is correct, but this might need revision if the percentages are different for each animal. In contrast, in Results, line 6, and in the discussion (third paragraph) 62% identity is reported. The authors should revise this inconsistency. Discussion, line 4 of third paragraph: “high percentage of identity” is better than “high conservation state”.\n\nIn the introduction, line 20: “Among patients allergic to rat, 73% and 87% reacted to Rat n 1 in dust”. Not clear why there are two percentages in this sentence. Is some information missing?\n\nLine 23-24: The reference above Böcskei et al. Nature 1992 should be added to the explanation of the structure of Rat n 1. Similarly, under methods section: “Construction of 3D models”, the pdb codes of the structures should be provided.\n\nIn Table 3: The abbreviations are not clear: If LE is discontinuous epitope (table title), then why in Figure 3 LE are linear epitopes? Are TCE, T-cell epitopes? Please define abbreviations.\n\nThe proper allergen nomenclature should be used to name lipocalins from hamster: Phod s 1, Mes a 1 (instead of Pho 21kD) in page 8 (discussion, second paragraph). Nomenclature can be found in the official WHO/IUIS Allergen Nomenclature database (www.allergen.org).\n\nBet v 1 is not a lipocalin family member as indicated in paragraph 4 of the discussion. This needs to be corrected.\n\nMinor comments:\nTable 1: species names should be in italics\n\nPage 5, line 11, “…constituted by 10 amino acid residues…”\n\nLegend to Figure 5: “discontinuous epitopes” instead of “epitopes discontinues”.\n\nDiscussion: second paragraph: “lipocalin family” instead of “lipocal infamily”.\n\nParagraph 4 of discussion: use of “enormous” should be avoided.\n\nBefore last paragraph of discussion: “This is the first exploration of the capacity of Rat n 1 to bind pheromones”. This is an in silico assay, and the study mentioned above (Böcskei et al) proved pheromone binding by X-ray crystallography.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "5054", "date": "12 Dec 2019", "name": "Marlon Munera", "role": "Author Response", "response": "Dear, Reviewer. We have taken note of all your comments. 1. The sentence in page 9: “Of the lipocalin family, only Mus m 1 has been characterized for pheromone linking” needs correction.R. Correction was made. 2. Ref: The major cockroach allergen Bla g 4 binds tyramine and octopamine. Offermann LR, Chan SL, Osinski T, Tan YW, Chew FT, Sivaraman J, Mok YK, Minor W, Chruszcz M. Mol Immunol. 2014 Jul;60(1):86-94R. Reference suggested was added.3. It would be good if the in silico analysis could be related to experimentally identified epitopes, and to discuss the results in the context of previous studies.R. All references suggested were incorporated 4. The rationale for selecting Can f 6 for the study should be explained since there are other dog lipocalins (Can f 1, Can f 2, Can f 4).R. Can f 6 was selected because share a moderated identity level with Rat n 1 and its 3D structure is resolved experimentally. 5-10. Abstract-resultsAll grammar advice and corrections were incorporated. On behalf of all authors, we appreciate all your comments to improve our research. ." } ] } ]	1	https://f1000research.com/articles/8-1707
https://f1000research.com/articles/7-1977/v1	24 Dec 18	{ "type": "Research Article", "title": "Phytochemical and antioxidant activity evaluation of the bark of Tampoi (Baccaurea macrocarpa)", "authors": [ "Erwin Erwin", "Widar Ristiyani Pusparohmana", "Indah Permata Sari", "Rita Hairani", "Usman Usman", "Widar Ristiyani Pusparohmana", "Indah Permata Sari", "Rita Hairani", "Usman Usman" ], "abstract": "Background: Tampoi (Baccaurea macrocarpa) is a tropical rainforest plant that produces edible fruit and is native to Southeast Asia, especially East Kalimantan, Indonesia. Previous research showed that Tampoi potentially can be developed as a drug. It was reported that the extract of Tampoi fruit displayed antioxidant activity, which was correlated with its phenolic and flavonoid substances. There is no information about the antioxidant activity of other parts of this plant, such as the bark, which might also have this kind of activity. Therefore, the aim of this study was to evaluate the phytochemical, toxicity, and antioxidant activity of the bark of Tampoi. Methods: The bark of Tampoi was extracted with methanol and concentrated using rotary evaporator to obtain the methanol extract of the bark. Secondary metabolites of this extract was determined using phytochemical analysis. Afterward, the methanol extract was tested for its toxicity using brine shrimp lethality test and antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl method. Results: Phytochemical evaluation results showed that the methanol extract of bark of this plant contains several secondary metabolites including alkaloids, flavonoids, phenolics, steroids, and triterpenoids. The toxicity test displayed no toxic property due to a LC50 value above 1000 ppm. For antioxidant activity, the result exhibited that the methanol extract of bark of this plant could be categorized as an active extract with IC50 value of 11.15 ppm. Moreover, based on gas chromatography-mass spectrometer analysis, there are 37 isolated compounds from the bark, one of which is methylparaben, a phenolic predicted to act as an antioxidant. Conclusion: The results obtained in this research demonstrated that the bark of Tampoi (B. macrocarpa) has potential as an antioxidant.", "keywords": [ "Tampoi", "Baccaurea macrocarpa", "toxicity", "BSLT", "antioxidant", "DPPH" ], "content": "Introduction\n\nIndonesia is a mega-diverse country in terms of biodiversity that is flanked by the Indian and Pacific Oceans. Indonesia's biodiversity encompasses the diversity of living things both on land and sea1. Indonesia, especially East Kalimantan, has very extensive tropical rainforest, which is a habitat for much biodiversity. Various types of plants have long been utilized by the community as traditional medicines. The utilization of natural products as an alternative medicine is increasing because natural ingredients are believed to be safer than synthetic substances, i.e. do not contain chemicals that only can be found in modern medicines, which are linked to toxicity2.\n\nAmong plants, the genus of Baccaurea have interesting biological activities. For example, B. angulata has been reported as a potential functional food with effective antioxidant3, anti-inflammatory, anti-atherogenic, and hypocholesteromia activities4. Other research has also investigated the biological activity of other species of this genus, i.e. B. lanceolata and B. macrocarpa. It was reported that the fruits of B. macrocarpa exhibited the highest antioxidant activity compared with B. lanceolata, which significantly correlated with the phenolic and flavonoid contents5.\n\nB. macrocarpa is one of the typical plants of East Kalimantan, Indonesia and the edible fruits is a source of additional nutrients and known as Tampoi. Until now, the information about the antioxidant activity of other parts of this plant such as the bark of Tampoi has not been reported yet. Hence, the present research was conducted to investigate the phytochemical, toxicity, and antioxidant activity of the bark of Tampoi (B. macrocarpa). Furthermore, the gas chromatography-mass spectrometer (GC-MS) analysis was performed to obtain information about the kinds of isolated compounds contained.\n\n\nMethods\n\nExtraction was carried out as described previously by Erwin et al. (2014)6. The bark of Tampoi (B. macrocarpa) was dried for 1 week at room temperature and ground to a powder. The powder was extracted using a maceration method by soaking in methanol for 24 hours at room temperature, which was repeated three times. Afterwards, the extract solution was filtered by filter paper and the solvent was evaporated under vacuum using a rotary evaporator (Buchi R II) at 45°C and 1 atm, to obtain the methanol extract of bark of Tampoi.\n\nPhytochemical evaluation was performed to investigate the secondary metabolites contents of the methanol extract of bark of Tampoi (B. macrocarpa), including alkaloids, flavonoids, phenolics, steroids, triterpenoids, and saponins, as described previously7. The presence of secondary metabolites were identified by observing the changing color of the extract. These evaluations were performed as follows:\n\nAlkaloids. 1 mg of extract was inserted into a test tube and then diluted in 1 mL methanol. Then a few drops of H2SO4 1M was added. Afterwards, a few drops of Dragendorff reagent was added into the mixture. The formation of orange on filter paper indicated the presence of alkaloids.\n\nFlavonoids. 1 mg of extract was inserted into a test tube and diluted in 1 mL methanol. A few 2 mg of Magnesium powder was added followed by a few drops of concentrated HCl. The presence of flavonoids was identified by the formation of pink or red color.\n\nPhenolics. 1 mg of extract was introduced into a test tube and dissolved in methanol. Then a few drops of 1% FeCl3 were inserted. The formation of green, red, purple, dark blue or black indicated the presence of phenolics.\n\nSteroids and triterpenoids. 1 mL of methanol and 1 mg of extract were inserted into a test tube, stirred until homogeneous, then 2 drops of anhydride acetate and 1 drop of H2SO4 were added (Liebermann Burchard reagent). The formation of green or purple precipitation showed a sample containing steroids, and red precipitation displayed the presence of terpenoids.\n\nSaponins. 1 mg extract was put into a test tube and then dissolved in distilled water, and shaken strongly. The presence of saponins is characterized by the formation of durable foam on the surface of the liquid. Foam that remains stable after the addition of a few drops of concentrated HCl indicated the presence of saponins.\n\nThe toxicity test of extract was performed using brine shrimp lethality test (BSLT), as described previously8. Methanol extract of bark of Tampoi (B. macrocarpa) (1 mg) was dissolved using 100 µL of 1% DMSO (dimethyl sulfoxide) and homogenized. The samples were diluted using 150 µL of distilled water until the total of volume reached 250 µL, and then pipetted 200 µL and diluted again using 600 µL of distilled water until the total of volume was 800 µL, so that the sample concentration was 1000 ppm. Samples with a concentration of 500, 250, 125, 62.5, 31.2, 15.6, and 7.8 ppm were made from sample dilutions of a concentration of 1000 ppm. The control solution was made with the same treatment as the sample without the addition of extract.\n\nThe toxicity test was carried out using several standard micro plates. About 100 µL seawater containing 8-13 shrimp larvae was added to each diluted sample so that the sample volume was 200 µL (with a concentration of 500, 250, 125, 62.5, 31.2, 15.6, and 7.8 ppm). The number of dead shrimp larvae was calculated for 24 hours after treatment. Each sample was treated in triplicate. The data obtained was recorded and the value of LC50 calculated (Lethal Concentration 50%) using SAS Probit analysis.\n\nThe antioxidant activity of the extract was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method, as described previously7,9–11. Briefly, the extract of bark of Tampoi (B. macrocarpa) was prepared in a solution with a concentration of 25, 50, 75 and 100 ppm, respectively. 1 mL of extract and 1 mL of DPPH (0.024 mg/mL) were put into a test tube, which was incubated for 30 min at 37°C before being measured by Spectrophotometer UV Thermo Scientific Evolution 201 (measurements were carried out at a wavelength of 515 nm). Vitamin C was used as a positive control with variations in concentration were 2, 4, 6, and 8 ppm, respectively. Determination of antioxidant activity or DPPH scavenging effect (%) of extract and vitamin C were carried out in triplicate using equation as follow.\n\n\n\nThen, the value of IC50 (Inhibitory Concentration 50%) was determined using linear regression.\n\nIn order to obtain the information of the kinds of compounds in methanol extract of bark of Tampoi, an analysis using GC-MS 5977 was performed. Specification of column that used in this research was HP-5MS with length 30 m, diameter 0.25 mm, thick of film 0.25 µm. The identification of the compound was compared to NIST standard data (https://webbook.nist.gov).\n\n\nResults\n\nThe secondary metabolites found in the methanol extract of the bark of Tampoi (B. macrocarpa) are presented in Table 1.\n\n(+): Presence; (-): Absence\n\nTo evaluate the antioxidant activity of the methanol extract of the bark, DPPH method was performed. The results of the antioxidant test can be seen in Table 2.\n\nAverage of three replicates performed for each concentration.\n\nFurthermore, the methanol extract was analyzed using GC-MS analysis. The chromatogram and it compound contents of this extract is shown in Figure 1 and Table 3, respectively.\n\n\nDiscussion\n\nBased on the phytochemical evaluation, the results showed that the methanol extract of bark of Tampoi (B. macrocarpa) contains several secondary metabolites including alkaloids, flavonoids, phenolics, steroids, and triterpenoids. Several secondary metabolites including alkaloids, steroids, triterpenoids, flavonoids, and phenolics are known to have antioxidant properties. These antioxidant compounds wield their activities through different mechanisms, for example by inhibiting hydrogen abstraction, radical scavenging, binding transition metal ions, disintegrating peroxides12,13, and one of the most important factors influencing antioxidant activity is the ability of the compounds to donate electrons.\n\nFurthermore, in the present study the antioxidant activity of the Tampoi extract was determined by DPPH method. This method was used because it is simple, efficient, quick, more practical, and relatively inexpensive14. Based on Table 2, it is known that the methanol extract of bark of Tampoi (B. macrocarpa) can be categorized as an active extract in an antioxidant assay with IC50 value of 11.15 ppm. In addition, the results of the toxicity test using the BSLT method showed that the extract was toxic because it displayed LC50 value above 1000 ppm.\n\nAccording to the results of GC-MS analysis, the chromatogram showed 37 peaks (compounds). The profile of the compounds showed that the main components were fatty acids and fatty acid esters. Total content of unsaturated fatty acids and esters with a peak area of 12.15% including 9,12-octadecadienoic acid (Z,Z)-, methyl ester (peak area 0.04), 9-octadecenoic acid, methyl ester (peak area 8.46), undec-10-ynoic acid, undecyl ester (peak area 3.58), undec-10-ynoic acid, undecyl ester (peak area 3.346), cis-vaccenic acid (peak area 0.07), and oleic acid (peak area 0.19). It was been reported that unsaturated fatty acid compounds and unsaturated fatty acid esters have significant antioxidant properties15–17.\n\nIt can be seen that only a small part of those are aromatic compounds. However, aromatic compounds are compounds that have the ability to stabilize high free radicals. The mechanism of phenolics as antioxidants is started by the formation a bond between free radical (DPPH radical) and hydrogen atom from OH-phenolics (ArOH) to form ArO. radical. Hydrogen atom will easier to be released because of the presence of electron withdrawing group which is bound at ortho- or para- positions18. Furthermore, ArO will react with a radical (ArO. or other radical) to form a stable compound19,20.\n\nDPPH. + AOH → DPPH-H + ArO.\n\nDPPH. + ArO. → DPPH-OAr or DPPH. + R. → DPPH-R\n\nAccording to identification of the compound in the methanol extract of bark of Tampoi (B. macrocarpa) using NIST database (DRUGBANK accession number, DB14212), it is known that the compound is identified as methylparaben. Based on the NIST database, peak at retention time at 9.467 min and peak area of 0.76% showed the characteristic of methylparaben (Molecular formula=C8H8O3; Molecular weight=152).\n\nIt has been reported that methylparaben does not show negative effects on male mouse reproduction21. Methylparaben is widely used as a preservative in cosmetic products, medicines or pharmaceutical products and food ingredients22,23, and the antibacterial activity of methylparaben is stronger than benzoate acid24.\n\nMethylparaben is a phenolic group that can reduce free radicals because it contains aromatic groups, -OH clusters and carbonyl groups. The presence of –COOCH3 substituent at para- position in methylparaben makes this compound act as an electron withdrawing group. The bond dissociation energy (BDE) of the O–H bond is a main factor to investigate the action of antioxidant, due to the weaker OH bond the reaction of the free radical will be easier19. As the prediction of the previous reaction mechanism7,19, the prediction of the reaction mechanism between DPPH radical and methyl paraben can be seen in Figure 2.\n\n\nConclusion\n\nThe results of the study showed that the bark of Tampoi (Baccaurea macrocarpa) has antioxidant activity with an IC50 value of 11.15 ppm.\n\n\nData availability\n\nF1000Research: Dataset 1. Sheet 1, raw data of the results of phytochemical evaluation for alkaloids, flavonoids, phenolics, steroids, triterpenoids, and saponins by observing the changing of colors; Sheet 2, raw data of the observation of the mortality numbers of Artemia salina Leach and calculation of LC50 value in toxicity test using brine shrimp lethality test; Sheet 3, raw data for antioxidant activity by DPPH method, including the measurement of absorbance using spectrophotometer in triplicate, the calculation of percentage of antioxidant activity, and the value of IC50; Sheet 4, raw data of GC-MS analysis., https://doi.org/10.5256/f1000research.16643.d22722225", "appendix": "Grant information\n\nThe authors acknowledge funding from the Islamic Development Bank (IsDB) project in the frame of Hibah Penelitian PIU IDB for Lecturer Mulawarman University 2018 Number: 2248/UN17.11/PL/2018.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors would like to thank the IsDB project for providing financial support and Head of Plant Anatomy and Systematic Laboratory of Biology Department, Faculty of Mathematics and Natural Sciences of Mulawarman University for identification the specimen.\n\n\nReferences\n\nYuwono A: The Fifth National Report of Indonesia to the Convention on Biological Diversity. Ministry of Environment and Forestry of Indonesia. 2014. Reference Source\n\nHussin AHJ: Adverse Effect of Herbs and Drug-Herbal Interactions. Malaysian Journal of Pharmacy. 2001; 1(2): 39–44. Reference Source\n\nIbrahim D, Hazali N, Jauhari N, et al.: Physicochemical and Antioxidant Characteristics of Baccaurea angulata Fruit Juice Extract. Afr J Biotechnol. 2013; 12(34): 5333–5338. Publisher Full Text\n\nIbrahim M, Ahmed IA, Mikail MA, et al.: Baccaurea angulata fruit juice reduces atherosclerotic lesions in diet-induced Hypercholesterolemic rabbits. Lipids Health Dis. 2017; 16(1): 134. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBakar MF, Ahmad AN, Karim FA, et al.: Phytochemicals and Antioxidative Properties of Borneo Indigenous Liposu (Baccaurea lanceolata) and Tampoi (Baccaurea macrocarpa) Fruits. Antioxidants (Basel). 2014; 3(3): 516–525. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nErwin, Noor A, Soekamto NH, et al.: Waltherione C and Cleomiscosin from Melochia umbellata var. Degrabrata K. (Malvaceae), Biosynthetic and Chemotaxonomic Significance. Biochem Syst Ecol. 2014; 55: 358–361. Publisher Full Text\n\nErwin, Nisa RN, Daniel: Phytochemical Test, Toxicity and Antioxidant Activity Leaves Kerehau (Callicarpa longifolia Lam.) with DPPH Method. Indonesia Chimica Acta. 2015; 8(1): 52–59. Reference Source\n\nMeyer BN, Ferrighi NR, Putnam JE, et al.: Brine shrimp: a convenient general bioassay for active plant constituents. Planta Med. 1982; 45(5): 31–34. PubMed Abstract \| Publisher Full Text\n\nErwin: Phytocemical Analysis and Antioxidant Activity of the Wood Ethanolic Extract of Sirih Hutan (Piper aduncum). Indonesia Chimica Acta. 2015; 8(20): 11–16. Reference Source\n\nAnokwah D, Mensah AY, Amponsah IK, et al.: Anti-inflammatory, Antioxidant and Antimicrobial Activities of the Stem Bark of Psydrax subcordata. Der Pharmacia Lettre. 2016; 8(20): 21–28. Reference Source\n\nBorkataky M: Antioxidant Activity, Total Phenolic Content and Total Flavonoid Content of Perilla ocymoides Linn. Der Pharmacia Lettre. 2015; 7(5): 69–72. Reference Source\n\nDiplock AT: Will the 'good fairies' please prove to us that vitamin E lessens human degenerative disease? Free Radic Res. 1997; 27(5): 511–532. PubMed Abstract \| Publisher Full Text\n\nPrior RL, Wu X, Schaich K: Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem. 2005; 53(10): 4290–4302. PubMed Abstract \| Publisher Full Text\n\nAkar Z, Küçük M, Doğan H: A new colorimetric DPPH• scavenging activity method with no need for a spectrophotometer applied on synthetic and natural antioxidants and medicinal herbs. J Enzyme Inhib Med Chem. 2017; 32(1): 640–647. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElagbar ZA, Naik RR, Shakya AK, et al.: Fatty Acids Analysis, Antioxidant and Biological Activity of Fixed Oil of Annona muricata L. Seeds. J Chem. 2016; 1–6, 6948098. Publisher Full Text\n\nPerona JS, Archemis C, Ruiz-Gutierrez V, et al.: Effect of dietary high-oleic-acid oils that are rich in antioxidants on microsomal lipid peroxidation in rats. J Agric Food Chem. 2005; 53(3): 730–735. PubMed Abstract \| Publisher Full Text\n\nPintato MEA, Araújo SG, Morais MI, et al.: Antifungal and antioxidant activity of fatty acid methyl esters from vegetable oils. An Acad Bras Cienc. 2017; 89(3): 1671–1681. PubMed Abstract \| Publisher Full Text\n\nBendary E, Francis RR, Ali HMG, et al.: Antioxidant and Structure–Activity Relationships (SARs) of Some Phenolic and Anilines Compounds. Annals of Agricultural Science. 2013; 58(2): 173–181. Publisher Full Text\n\nBrand-Willians W, Cuvelier ME, Berset C: Use of a free radical Method to Evaluate Antioxidant Activity. LWT - Food Sci Technol. 1995; 28(1): 25–30. Publisher Full Text\n\nVillaño D, Fernández-Pachón MS, Moyá ML, et al.: Radical scavenging ability of polyphenolic compounds towards DPPH free radical. Talanta. 2007; 71(1): 230–235. PubMed Abstract \| Publisher Full Text\n\nCommittee for Medicinal Products for Human Use (CHMP): Reflection Paper on the Use of Methyl- and PropylParaben as Excipients in Human Medicinal Products for Oral Use. European Medicines Agency Science Medicines Health. 2015; 1–13. Reference Source\n\nMacy E, Schatz M, Zeiger RS: Immediate Hypersensitivity to Methylparaben Causing False-Positive Results of Local Anesthetic Skin Testing or Provocative Dose Testing. Perm J. 2002; 6(4): 17–21. Free Full Text\n\nMicea MM, Lupşa IR, Cinghiţă DF, et al.: Determination of Methylparaben from Cosmetic Products by Ultra Performance Liquid Chromatography. J Serb Chem Soc. 2009; 74(6): 669–676. Publisher Full Text\n\nMirsonbol SZ, Issazadeh K, Pahlaviani MRMK, et al.: Antimicrobial Efficacy of the Methylparaben and Benzoate Sodium against Selected Standard Microorganisms, Clinical and Environmental Isolates In Vitro. Indian Journal of Fundamental and Applied Life Sciences. 2014; 4(S4): 363–367. Reference Source\n\nErwin E, Pusparohmana WR, Sari IP, et al.: Dataset 1 in: Phytochemical and antioxidant activity evaluation of the bark of Tampoi (Baccaurea macrocarpa). F1000Research. 2018. http://www.doi.org/10.5256/f1000research.16643.d227222" }	[ { "id": "44034", "date": "04 Nov 2019", "name": "Chinnadurai Immanuel Selvaraj", "expertise": [ "Reviewer Expertise Plant Phyto chemistry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study design and the methodology sounds good. Still more details on the plant B. macrocarpa need to be included in the introduction. Even though the authors try to substantiate Methyl paraben as a non-toxic compound, it is a universally known fact usage of Methyl paraben is strongly discouraged in all human usage food and cosmetics as preservative. The Environmental Working Group (EWG) lists methylparaben as being a low to moderate Health Hazard. Parabens are potential endocrine disruptors due to their ability to mimic estrogen. Studies demonstrate that at sufficient concentrations, parabens can increase cell proliferation in human breast cancer MCF-7 cells, which are often used as a sensitive measure of estrogenic activity. Applying personal care product containing parabens—especially methylparaben—can lead to UV-induced damage of skin cells and disruption of cell proliferation (cell growth rate). These are evidenced reports on the Methyl paraben. Nevertheless, it is available in the natural source from the plant in meagre quantity. The authors can check for other compounds in GC-MS and state its importance in the manuscript. The GC-MS can be repeated. or HPLC can be performed using an aqueous extract. A simple TLC becomes handy for compound prediction, Then a column chromatography will be useful to check if there are any useful compounds.\nThe authors fail to include the ill effects of Methyl paraben in the literature. Is there any reason for avoiding such inclusions? The authors should weigh the importance of other compounds in the plant. Is there any traditional/ancient usage of the fruit mentioned in literature must be included in Introduction section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5037", "date": "12 Dec 2019", "name": "erwin erwin", "role": "Author Response", "response": "Reviewer comment #1The authors can check for other compounds in GC-MS and state its importance in the manuscriptAuthor response #1We have re-checked the 37 peaks which appeared to be methylparaben most likely to be antioxidants Reviewer comment #2The GC-MS can be repeated or HPLC can be performed using an aqueous extract. A simple TLC becomes handy for compound prediction, Then a column chromatography will be useful to check if there are any useful compounds.Author response #2This study is an initial screening of the antioxidant activity of Tampoi bark. The next project if we get the funding, we will use other instruments as you suggested. We will also carry out isolation and purification to obtain active compounds from Tampoi bark. We appreciated your suggestionReviewer comment #3The authors fail to include the ill effects of Methyl paraben in the literatureAuthor response #3We have added additional literature about the side effects of methyl paraben. Ref. no: 27Reviewer comment #4Is there any traditional/ancient usage of the fruit mentioned in literature must be included in the Introduction sectionAuthor response #4We did not find any use of tampoi as traditional medicine, but several other species of the genus Baccuarea were used as traditional medicine. In addition, there are several preliminary studies on Tampoi's bio-activity. Ref. no: 3,4,8, and 9" } ] }, { "id": "56217", "date": "06 Nov 2019", "name": "Natthida Weerapreeyakul", "expertise": [ "Reviewer Expertise Pharmacology", "Biomedical sciences" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAccording to the GC chromatogram the peak that was claimed to be methyl paraben was detected at 9.479 min but when identified with the MS the peak at 9.467 min was identified instead. This might be wrong interpretation. Why the peak with high intensity detected at 19.329 min of methyl palmitate was not considered as the major compound or whether it was contributed to the antioxidant effect? Due to there are many antioxidant mechanisms, therefore, only DPPH scavenging activity is not sufficient.\n\nCytotoxicity result was not shown and sufficiently discussed in correlation with the antioxidant activity. The statistical analysis should be mentioned in the method section. Based on the insufficient information and evidence, this manuscript needs more experimentation and well written regarding the method, results and discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "56219", "date": "11 Nov 2019", "name": "Agustono Wibowo", "expertise": [ "Reviewer Expertise Natural Product Chemistry and Organic Synthesis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIntroduction:\nIn first paragraph line 5, please correct your statement on natural ingredients “do not contain chemicals” that only can be found in modern medicines, as all things in this world is formed from chemical constituents. Please change to “contain toxic chemicals”.\n\nLast paragraph line 5, the statement “kinds of isolated compounds contained” are not correct as you don’t isolate the compound. Please remove the word “isolated”.\nMethods:\nDDPH assay alone can’t express the antioxidant properties of sample, so we suggest you to add other antioxidant assay such as ABTS and FRAP.\nDiscussion:\nGCMS result indicated that the main constituent in Baccaurea macrocarpa extract is fatty acid, this is because the GCMS can only detect the volatile compounds. To identify other compounds that are responsible in the antioxidant activity of Baccaurea macrocarpa, we suggest you to run your sample using LCMS.\n\nMethylparaben is familiar compound. Can you give literature which supported your claim that methylparaben is responsible to the antioxidant of Baccaurea macrocarpa extract?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5038", "date": "12 Dec 2019", "name": "erwin erwin", "role": "Author Response", "response": "Reviewer comments #1In first paragraph line 5, please correct your statement on natural ingredients “do not contain chemicals” that only can be found in modern medicines, as all things in this world is formed from chemical constituents. Please change to “contain toxic chemicals”Author response #1Thank you for your suggestion, It has been fixedReviewer comments #2The statement “kinds of isolated compounds contained” are not correct as you don’t isolate the compound. Please remove the word “isolated”Author response #2The word isolated has been removedReviewer comments #3Methods:DPPH assay alone can’t express the antioxidant properties of the sample, so we suggest you add other antioxidant assays as ABTS and FRAPAuthor response #3This study is an initial screening of the antioxidant activity of Tampoi bark.Reviewer comments #4Discussion:GCMS result indicated that the main constituent in Baccaurea macrocarpa extract is a fatty acid, this is because the GCMS can only detect the volatile compounds. To identify other compounds that are responsible for the antioxidant activity of Baccaurea macrocarpa, we suggest you to run your sample using LCMS.Author response #4The next project if we get the funding, we will use another antioxidant test and other instruments as you suggest. We will also carry out isolation and purification to obtain active compounds from Tampoi bark.Reviewer comments #5Methylparaben is a familiar compound. Can you give literature which supported your claim that methylparaben is responsible for the antioxidant of Baccaurea macrocarpa extract?Author response #5I could not find in the literature that discusses the antioxidant properties of parabens in vegetation, but methylparaben is preservative and antioxidant in cosmetic products, medicines or pharmaceutical products, and food ingredients. Based on GC-MS data, methylparaben is most likely to be antioxidants" } ] }, { "id": "56216", "date": "15 Nov 2019", "name": "Chanya Chaicharoenpong", "expertise": [ "Reviewer Expertise Natural Product Chemistry." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript reported the new information of phytochemical and antioxidant activity of barks of Baccaurea macrocarpa.\nThe authors used only DPPH assay to evaluate antioxidant activity. Antioxidant activity should be investigated using various assays to present antioxidant capacity of the extracts.\nThe results of evaluation on phytochemicals of barks of B. macrocarpa showed that the methanol extract consisted of alkaloids, steroids, triterpenoids, flavonoids and phenolic compounds. But the profile of GC-MS showed only fatty acids, fatty acid esters and methyl paraben. The authors should use LC-MS to investigate chemical constituents of methanol extract instead of GC-MS.\nIn results section, the authors did not mention on the toxicity test of the extract using brine shrimp lethality test. And the results should express yours statistical analysis.\nIn discussion section, the third paragraph, the authors need to rewrite the total content and composition of fatty acids. GC chromatogram in Figure 1 was not related to the data of composition of compounds in Table 3 such as retention time, % peak area. For example, peak at retention time 19.329 showed high intensity on GC chromatogram but it expressed low % peak area just 0.91. The peak at retention time 14.877 showed low intensity on GC chromatogram but it expressed % peak area 1.32. Moreover, some compounds presented low matching percentage from the library searching. In Figure 2, structure of methyl paraben was wrong.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5039", "date": "12 Dec 2019", "name": "erwin erwin", "role": "Author Response", "response": "Reviewer comment #1The authors used only DPPH assay to evaluate antioxidant activity. The antioxidant activity should be investigated using various assays to present antioxidant capacity of the extracts. The results of evaluation on phytochemical of barks of B. macrocarpa showed that the methanol extract consisted of alkaloids, steroids, triterpenoids, flavonoids and phenolic compounds. But the profile of GC-MS showed only fatty acids, fatty acid esters and methyl paraben. The authors should use LC-MS to investigate chemical constituents of methanol extract instead of GC-MS.Author response #1This study is an initial screening of the antioxidant activity of Tampoi bark. The next project if we get the funding, we will use another antioxidant test and other instruments as you suggest. We will also carry out isolation and purification to obtain active compounds from Tampoi bark. Thank you for your valuable suggestionReviewer comment #2In the results section, the authors did not mention on the toxicity test of the extract using brine shrimp lethality test. And the results should express your statistical analysisAuthor response #2Toxicity test data have been added in the revised articleReviewer comment #3some compounds presented low matching percentage from the library searchingAuthor response #3It has been fixedReviewer comment #4In Figure 2, the structure of methylparaben was wrong.Author response #4The structure of methylparaben has been fixedReviewer comment #5In the discussion section, the third paragraph, the authors need to rewrite the total content and composition of fatty acids. GC chromatogram in Figure 1 was not related to the data of composition of compounds in Table 3 such as retention time, % peak area. For example, peak at retention time 19.329 showed high intensity on GC chromatogram but it expressed a low % peak area just 0.91. The peak at retention time 14.877 showed low intensity on GC chromatogram but it expressed % peak area 1.32.Author response #5The percentage (%) peak area of GC chromatogram has been fixed" } ] } ]	1	https://f1000research.com/articles/7-1977
https://f1000research.com/articles/8-2093/v1	12 Dec 19	{ "type": "Research Article", "title": "Invariance in ecological pattern", "authors": [ "Steven A. Frank", "Jordi Bascompte", "Jordi Bascompte" ], "abstract": "Background: The abundance of different species in a community often follows the log series distribution. Other ecological patterns also have simple forms. Why does the complexity and variability of ecological systems reduce to such simplicity? Common answers include maximum entropy, neutrality, and convergent outcome from different underlying biological processes. Methods: This article proposes a more general answer based on the concept of invariance, the property by which a pattern remains the same after transformation. Invariance has a long tradition in physics. For example, general relativity emphasizes the need for the equations describing the laws of physics to have the same form in all frames of reference. Results: By bringing this unifying invariance approach into ecology, we show that the log series pattern dominates when the consequences of processes acting on abundance are invariant to the addition or multiplication of abundance by a constant. The lognormal pattern dominates when the processes acting on net species growth rate obey rotational invariance (symmetry) with respect to the summing up of the individual component processes. Conclusions: Recognizing how these invariances connect pattern to process leads to a synthesis of previous approaches. First, invariance provides a simpler and more fundamental maximum entropy derivation of the log series distribution. Second, invariance provides a simple derivation of the key result from neutral theory: the log series at the metacommunity scale and a clearer form of the skewed lognormal at the local community scale. The invariance expressions are easy to understand because they uniquely describe the basic underlying components that shape pattern.", "keywords": [ "Macroecology", "neutral theory", "maximum entropy", "symmetry" ], "content": "\n\n“It was Einstein who radically changed the way people thought about nature, moving away from the mechanical viewpoint of the nineteenth century toward the elegant contemplation of the underlying symmetry [invariance] principles of the laws of physics in the twentieth century” (ref. 1, p. 153).\n\n\nIntroduction\n\nEcologists have been interested in species abundance distributions (SADs) since the classic papers by Fisher2 and Preston3. Two major patterns have been identified depending on the size of the community. In a large community, abundances often follow the log series distribution4. Specifically, the probability that a species has a population size of n individuals follows pn/n. Communities differ only in their average population size, described by the parameter, p. At smaller spatial scales, the species abundance pattern often follows a skewed lognormal (a random variable is lognormally distributed when its logarithm is normally distributed)5,6.\n\nIt is intriguing that the species abundance distribution follows these simple patterns irrespective of the particular group (birds, insects, mammals) and region considered. Other ecological patterns also follow simple probability distributions7–9. Those patterns have attracted a lot of attention. Why does the variability and complexity of biology reduce to such a small range of simple distributions? How can we understand the relations between complex processes and simple patterns?\n\nApproaches such as Harte’s9 maximum entropy formalism and Hubbell’s5 neutral theory have attempted to explain the generality of the log series and skewed lognormal patterns in species abundance distributions. Maximum entropy describes probability distributions that are maximally random subject to satisfying certain constraints10–12. This approach has a long tradition in physics, both in statistical mechanics and information theory. An early maximum entropy approach in ecology derived the biomass pattern of populations13–15.\n\nNeutral theory derives probability distributions by assuming that all individuals are equivalent16. Variation arises by random processes acting on the mechanistically identical individuals. Put another way, the mechanistic processes are “neutral” apart from random processes. Both maximum entropy and neutral theory have been shown to provide a good fit to the empirical patterns of species abundance distributions. In this article, we subsume these two different ways of understanding the log series and skewed lognormal patterns with a more general perspective based on the concept of invariance17.\n\nInvariance can be defined as the property by which a system remains unchanged under some transformation. For example, a circle is the same (invariant) before and after rotation (Figure 1a). In ecology, pattern often depends on the ways in which form remains invariant to changes in measurement. Some patterns retain the same form after uniformly stretching or shrinking the scale of measurement (Figure 2b). Measures of length provide a common example of stretch invariance. One can measure lengths equivalently in millimeters or centimeters without loss of information. As we will see, that kind of invariance often determines the form of observed pattern.\n\n(a) Transforming a circle by rotation leaves the circle unchanged (invariant), with an invariant radial distance at all points along the circumference. (b) Rotating regular polygons changes pattern. However, as more rotated polygons are added, the form converges asymptotically to a rotationally invariant circle, in which adding another rotated polygon does not change the pattern. Many common patterns of nature are asymptotically invariant. In this case, aggregation causes loss of all information except invariant radial distance. (c) The normal distribution is asymptotically invariant. The left curve describes an arbitrary probability pattern. The second curve expresses the sum of two randomly chosen values from the first curve. The height is the relative probability of the summed values. The third, fourth, and fifth curves express the sum of 4, 8, and 16 randomly chosen values from the first curve. Each curve width is shrunk to match the first curve. In this case, aggregation smooths the curve, causing loss of all information except the average squared distance from the center (the variance), which is equivalent to the average squared radial distance of rotationally invariant circles. (d) Extreme value distributions are asymptotically invariant. The left curve is an arbitrarily chosen probability pattern. The second curve expresses the probability of the largest value in a sample of two randomly chosen values from the first curve. The third, fourth, and fifth curves show the probability of the largest value of 4, 8, and 16 randomly chosen values. The asymptotically invariant curve on the right expresses exponential scaling at small values and linear scaling at large values, labeled in green and blue. Commonly observed probability distributions often express simple combinations of linear, logarithmic, and exponential scaling. Panels (a–c) modified from Frank17.\n\n(a) The left panel shows e–(x+a) for a = 0, –1, . . . , –4. Decreasing values of a shift the curve to the right, which is equivalent to shifting the x axis by resetting the zero point. For probability patterns, the total probability must be normalized to one, which means that all curves must have the same area under the curve for values of x between 0 and ∞. To normalize the curves, the right panel plots kae–(x +a) with ka = ea. Thus, all curves become e–x invariantly with respect to different shift values, a. (b) The left panel shows e–bx for b = 20, 2–1, . . . , 2–4. Decreasing values of b stretch the x axis by a factor of 2 for each halving of b. To normalize the average value of each probability curve to be the same, the right panel shows e–λbbx for λb = 1/b. Thus, all curves become e–x invariantly with respect to different stretch values, b.\n\nTo give another example, consider the common and widely familiar pattern of the normal distribution. By the central limit theorem, when independent random variables are added, their properly normalized sum tends toward a normal distribution, even when the component variables themselves are not normally distributed. The central limit theorem and the normal distribution are often considered as unique aspects of pattern that stand apart from other commonly observed patterns.\n\nThe invariance perspective that we promote shows how the normal distribution is in fact a specific example of a wider framework in which to understand the commonly observed patterns of nature. In particular, the normal distribution arises from the rotational invariance of the circle18. For two variables, x and y, with a given squared length, x2 + y2 = r2, all combinations of the variables with the same radius, r, lie along the circumference of a circle (Figure 1a). When each combination is equally likely, the rotationally invariant radius is sufficient to describe the probability pattern.\n\nIt is this rotational invariance that gives the particular mathematical form of the normal distribution, in which the average squared radius sets the variance of the distribution. By this perspective, the mathematical forms of all commonly observed distributional patterns express their unique invariances18.\n\nThe perspective of invariance was the basis for most of the great conceptual advances of physics in the twentieth century1. For example, Gell-Mann’s pioneering theoretical work on the fundamental particles of nature derived from invariance (symmetry) properties that unified understanding of known particles and predicted new particles such as quarks, which were subsequently observed. By contrast, general aspects of invariance have not been used consistently as the fundamental basis for understanding patterns in ecology. One exception concerns scale invariance, which is often discussed in ecology19–21. But scale invariance is typically limited to special kinds of patterns rather than forming a unified approach to diverse patterns.\n\nThe point of this paper is that invariance is the most general way in which to understand commonly observed patterns. Species abundance distributions provide an excellent illustration. For species abundances, we show that maximum entropy and neutral models can succeed in certain cases because they derive from invariance principles. However, maximum entropy and neutrality are often difficult to interpret because they hide their underlying basis in invariance.\n\nOur unifying invariance analysis clarifies why seemingly different conceptual approaches have the same consequences for pattern. Similarly, seemingly different biological processes may often lead to the same observed pattern, because those different processes share a common basis in invariance. That deeper understanding suggests a more insightful way to think about alternative mechanistic models. It also suggests the kinds of empirical tests that may differentiate between alternative causal processes.\n\nThis manuscript is organized as follows. First, we highlight key theoretical results for species abundance distributions. Second, we review how invariance defines probability patterns in a general way18,22,23. The log series distribution24 and the gamma-lognormal distribution for species abundances follow directly from the universal invariance expression of probability patterns. Third, we show that maximum entropy and neutrality can easily be understood as special examples of invariance principles. Finally, we discuss the broad role of invariance in the analysis of ecological pattern.\n\n\nKey results\n\nThis article develops two key theoretical results. We highlight those results before starting on the general overview of invariance and pattern.\n\nFirst, we present a simple maximum entropy derivation of the log series pattern. We show that constraining the average abundance per species is sufficient when analyzing randomness and entropy on the proper demographic scale25.\n\nThe simplicity of our maximum entropy derivation contrasts with Harte’s more complicated maximum entropy model9,26. Harte had to assume an additional unnecessary constraint on energy usage. He required that unnecessary constraint because he evaluated randomness on the scale of measured abundances rather than on the scale of demographic process. This will be made explicit below.\n\nWe use this result to demonstrate that maximum entropy is the outcome of deeper underlying principles of invariance and pattern. By working at the deeper level of invariance, one obtains a simpler and more powerful understanding of pattern.\n\nThe second result shows that Hubbell’s5 neutral model is the simple expression of three basic invariances. Hubbell’s full range of log series and skewed lognormal (zero sum multinomial) results follows immediately from those three underlying invariances.\n\nThe three invariances correspond to a maximum entropy model that constrains the average abundance of species and the average and variance of the demographic processes influencing abundance. The three invariances lead to a simple gamma-lognormal distribution that matches the neutral theory pattern for species abundances25. The gamma-lognormal is a product of the standard gamma and lognormal distributions.\n\n\nInvariance\n\nThis section reviews how invariance considerations lead to the log series distribution24. We delay discussion of the gamma-lognormal until the later section on Hubbell’s neutral model.\n\nWe can rewrite almost any probability distribution as\n\n\n\nin which T(z) ≡ Tz is a function of the variable, z, and k and λ are constants. For example, Student’s t-distribution, usually written as\n\n\n\ncan be written in the form of Equation 1 with λ = (ν + 1)/2 and Tz = log(1 + z2/ν).\n\nThe probability pattern, qz, is invariant to a constant shift, Tz ↦ a + Tz, because we can write the transformed probability pattern in Equation 1 as\n\n\n\nwith k = kae–λa (Figure 2a). We express k in this way because k adjusts to satisfy the constraint that the total probability be one. In other words, conserved total probability implies that the probability pattern is shift invariant with respect to Tz18.\n\nNow consider the consequences if the average of some value over the distribution qz is conserved. For example, the average of z is the mean, µ, and the average of (z – µ)2 is the variance. A constraint causes the probability pattern to be invariant to a multiplicative stretching (or shrinking), Tz ↦ bTz, because\n\n\n\nwith λ = λbb (Figure 2b). We specify λ in this way because λ adjusts to satisfy the constraint of conserved average value. Thus, invariant average value implies that the probability pattern is stretch invariant with respect to Tz.\n\nConserved total probability and conserved average value cause the probability pattern to be invariant to an affine transformation of the Tz scale, Tz ↦ a + bTz, in which “affine” means both shift and stretch.\n\nThe affine invariance of probability patterns with respect to Tz induces significant structure on the form of Tz and the associated form of probability patterns. Understanding that structure provides insight into probability patterns and the processes that generate them18,22,23.\n\nIn particular, Frank and Smith22 showed that the invariance of probability patterns to affine transformation, Tz ↦ a + bTz, implies that Tz satisfies the differential equation\n\n\n\nin which w(z) is a function of the variable z. The solution of this differential equation expresses the scaling of probability patterns in the generic form\n\n\n\nin which, because of the affine invariance of Tz, we have added and multiplied by constants to obtain a convenient form, with Tz → w as β → 0.\n\nBy writing Tz in this way, w expresses a purely shift-invariant aspect of the fundamental affine-invariant scale, because the shift transformation w ↦ a + w multiplies Tz by a constant, and probability pattern is invariant to constant multiplication of Tz. Thus, Equation 2 dissects the anatomy of a probability pattern (Equation 1) into its component invariances.\n\nWith this expression for Tz, we may write probability patterns generically as\n\n\n\nThis form has the advantage that w(z) expresses the shift-invariant structure of a probability pattern. Most of the commonly observed probability patterns have a simple form for w23,27. That simplicity of the shift-invariant scale suggests that focus on w provides insight into common patterns.\n\nTo understand the log series, we must consider the relation n = er between the observed pattern of abundances, n, and the processes, r. Here, r represents the total of all proportional processes acting on abundance24.\n\nA proportional process simply means that the number of individuals or entities affected by the process increases in proportion to the number currently present, n. Demographic processes, such as birth and death, act proportionally.\n\nThe sum of all of the proportional processes on abundance over some period of time is\n\n\n\nHere, m(t) is a proportional process acting at time t to change abundance. Birth and death typically occur as proportional processes. The value of r = log n is the total of the m values over the total time, τ. For simplicity, we assume n0 = 1.\n\nThe log series follows as a special case of the generic probability pattern in Equation 3. To analyze abundance, focus on the process scale by letting the variable of interest be z ≡ r, with the key shift-invariant scale as simply the process variable itself, w(r) = r. Then Equation 3 becomes\n\n\n\nin which qrdr is the probability of a process value, r, in the interval r + dr.\n\nUsing w(r) = r sets the the shift-invariant scale as the variable itself. Substituting this simplest form for the shift-invariant scale into the canonical equation for common probability patterns in Equation 3 yields the simplest generic expression of probability pattern as Equation 4.\n\nWe can generalize the relation between abundance and process, n = er, by writing nβ = eβr, which uses an additional parameter β to allow comparison with the canonical form of probability distributions in the previous subsection. When we focus on standard models of species abundances, we use β = 1.\n\nWe can change from the process scale, r, to the abundance scale, n, by noting that β log n = βr, and so, for any β, we have r = log n. Thus, we can use the substitutions r ↦ log n and dr ↦ n–1dn in Equation 4, yielding the identical probability pattern expressed on the abundance scale\n\n\n\nThe value of k always adjusts to satisfy the constraint of invariant total probability, and the value of λ always adjusts to satisfy the constraint of invariant average value.\n\nFor proportional processes and species abundances, β = 1, as noted above. For that value of β, we obtain the log series distribution24\n\n\n\nreplacing n – 1 by n in the exponential term which, because of affine invariance, describe the same probability pattern. The log series is often written with e–λ = p, and thus qn = kpn/n. One typically observes discrete values n = 1, 2, …. See https://doi.org/10.5281/zenodo.2597895 for the general relation between discrete and continuous distributions. The continuous analysis here is sufficient to understand pattern.\n\nWe can also write the log series on the process scale, r, from Equation 4, as 24\n\n\n\nThis form shows that the log series is the simplest expression of generic probability patterns in Equation 3. The log series arises from β = 1, associated with n = er, and from the base shift-invariant scale as w ≡ r for proportional processes, r.\n\nThis subsection summarizes a few technical points about invariance. These technical points provide background for our simpler and more general derivation in the following section of maximum entropy models for species abundances. Those previous models focused only on abundances, n, without considering the underlying process scale, r.\n\nWe begin with invariance on the process scale, r. On that scale, the log series in Equation 7 is the pure expression of additive shift invariance to r and lack of multiplicative stretch invariance to r. For example, note in Equation 7 that an additive change, r ↦ r + a, is compensated by a change in λ to maintain the overall invariance, whereas a multiplicative change, r ↦ br, cannot be compensated by a change in one of the constants. For example, if r is net reproductive rate, then an improvement in the environment that adds a constant to everyone’s reproductive rate does not alter the log series pattern. By contrast, multiplying reproductive rates by a constant does alter pattern.\n\nTo understand the parameter, β, from Equation 2, consider that\n\n\n\nin which β is the relative curvature of the measurement scale for abundance, n, with respect to the scale for process, r. The relative curvature is β = T″/T′, with the primes denoting differentiation with respect to r27.\n\nFor the log series, the curvature of β = 1 describes the amount of bending of the abundance scale, n = er, with respect to multiplying the process scale, r, by a constant—the departure from stretch invariance.\n\nThe simple invariances with respect to process, r, become distorted and more difficult to interpret when we focus only on the observed scale for abundance, n, associated with the log series in Equation 6. In that form of the distribution, the canonical scale is\n\n\n\nIn this expression, purely in terms of abundances, the log-arithmic term dominates when n is small, and the linear term dominates when n is large. Thus, the scale changes from stretch but not shift invariant at small magnitudes to both shift and stretch invariant at large magnitudes24. Without the simple insight provided by the process scale, r, we are left with a complicated and nonintuitive pattern that is separated from its simple cause. That difficulty has led to unnecessary complications in maximum entropy theories of pattern.\n\nPueyo et al. developed a simple alternative approach for deriving the log series distribution that combines invariance and maximum entropy25. In their derivation, the average value of n is a maximum entropy constraint, and the equivalent of our r variable is considered as an invariant Bayesian prior in the sense of Jaynes12. Previous publications describe the differences between our invariance approach and the invariant prior maximum entropy approach of Pueyo et al.18,22,28,29.\n\n\nMaximum entropy\n\nMaximum entropy describes probability distributions that are maximally random subject to satisfying certain constraints10–12. In Equation 1, with the generic description for distributions as\n\n\n\nmaximum entropy interprets this form as the expression of maximum randomness with respect to the scale z, subject to the constraint that the average of Tz is fixed23.\n\nThis section begins with a maximum entropy derivation for the log series based on our separation between the scales of process, r, and observed abundance, n.\n\nWe then discuss Harte’s9,26 alternative maximum entropy derivation of the log series. Harte’s derivation emphasizes mechanistic aspects of energy constraints rather than our emphasis on the different scales of process and abundance.\n\nThe log series in Equation 7 is\n\n\n\nHere, T = er = n. This distribution expresses maximum entropy with respect to the process scale, r. The constraint is the ecological limitation on average abundance\n\n\n\nin which 〈·〉r denotes average value with respect to the process scale, r.\n\nIn this case, process values, r, are maximally random, subject to the ecological constraint that limits abundance, n. Thus, maximizing entropy with respect to the process scale, r, subject to a constraint on the observed pattern scale, n, leads immediately to the log series.\n\nRelating the process scale, r, to the scale of ecological constraint, n, often makes sense. Typically, environmental perturbations associate with changes in demographic variables, such as birth and death rates. Such demographic factors typically act proportionally on populations, consistent with our interpretation of r as the aggregate of proportionally acting processes. The perturbations, acting on demographic variables, associate the process scale with the scale of randomness.\n\nIn contrast with the process scale of perturbation and randomness for the demographic variables, the scale of constraint naturally arises with respect to a limit on the number of individuals, n. Thus, randomness happens on the r scale and constraint happens on the n scale.\n\nIt is, of course, possible to formulate alternative models in which randomness and constraint happen on scales that differ from our interpretation. Different formulations are not intrinsically correct or incorrect. Instead, they express different assumptions about the relations between process, randomness, and invariance. The next section considers an alternative formulation.\n\nHarte developed comprehensive maximum entropy models of ecological pattern. He tested those theories with the available data. His work synthesizes many aspects of ecological pattern9.\n\nFor species abundances, Harte9,26 analyzed maximum randomness with respect to the scale of abundance values, n. Maximum entropy derivations commonly evaluate randomness on the same scale as the observations. In this case, with observations for the probabilities of abundances, pn, entropy on the same scale is the sum or integral of –pn log pn.\n\nHowever, there is no a priori reason to suppose that the scale of observation is the same as the scale of randomness. The fact that observation, randomness, and process may occur on different scales often makes maximum entropy models difficult to develop and difficult to interpret. For example, we may observe the probabilities of abundances, pn, but randomness may be maximized on the scale of process, as the sum or integral of –pr log pr.\n\nIn the final part of this section, we argue that invariance provides a truer path to the natural scale of analysis and to the mechanistic processes that generate pattern than does maximum entropy. Before comparing invariance and maximum entropy, it is useful to sketch the details of Harte’s maximum entropy model for species abundances.\n\nThe simplest maximum entropy model analyzes entropy with respect to abundance, n, subject to a constraint on the average abundance, ⟨n⟩. That analysis yields an exponential distribution\n\n\n\nThe exponential pattern differs significantly from the observed log series pattern. Thus, maximizing entropy with respect to the scale of abundance, n, and constraining the average abundance is not sufficient.\n\nFrom our invariance perspective, it is natural to think of the scale of randomness in terms of dr, the scale of proportional processes, rather than in terms of dn, the scale of abundance. Maximizing randomness with respect to dr leads directly to the log series, as shown in the previous section.\n\nHarte did not consider the distinction between the exponential and log series patterns with respect to the scale of randomness. Instead, to go from the default exponential pattern of maximum entropy to the log series, his maximum entropy analysis required additional assumptions. He proceeded in the following way.\n\nSuppose that the total quantity of some variable, 𝜖, is constrained to be constant over all individuals of all species. The average value per individual is ⟨𝜖⟩. It does not matter what the variable 𝜖 is. All that matters is that the constraint exists. Harte assumed that 𝜖 is energy, but that assumption is unnecessary with regard to the species abundance distribution.\n\nThe value 𝜖 is distributed over individuals independently of their species identity. Thus, the variable δ\|n = n𝜖 is the total value in a species with n individuals, with average value ⟨δ\|n⟩ = n⟨𝜖⟩.\n\nThe joint distribution of n and δ is\n\n\n\nThe explicit form of this joint distribution can be obtained by maximizing entropy subject to the constraints on the average abundance per species, ⟨n⟩, and the average total value in a species with n individuals, ⟨δ\|n⟩, yielding\n\n\n\nWe obtain the form presented by Harte26 using the equivalence δ = n𝜖, yielding\n\n\n\nThe species abundance distribution is obtained by\n\n\n\nNoting that ∫ e–λ′n𝜖 = 1/λ′n, and absorbing the constant λ′ into k, we obtain the log series for the species abunance distribution\n\n\n\nHarte’s maximum entropy derivation of the log series assumes joint constraints of abundance, n, and some auxiliary variable, 𝜖, which he labeled as energy. He evaluated entropy on the scales of n and 𝜖.\n\nBy contrast, our invariance derivation arises from a constraint on abundance plus evaluation of invariance or entropy on the scale r = log n. On that scale, the log series arises in a simple and clear way. There is no need for constraint of a second auxiliary variable.\n\nWithout an invariance argument, nothing compels us to analyze with respect to the r scale. Harte, without focus on invariance, followed the most natural approach of using n as the scale for maximization of randomness and for constraint. That approach required an auxiliary constraint on a second scale to arrive at the log series.\n\nHarte’s approach was a major step in unifying the analysis of empirical pattern. But, in retrospect, his approach was unnecessarily complicated.\n\nOne might say that Harte’s approach provided a richer theory because it led to predictions about both abundance and energy. However, the data on abundance patterns match very closely to the log series, whereas the data for different proxies of energy vary considerably9.\n\nOur invariance approach strips away the unnecessary auxiliary variable. The invariance theory therefore provides a much simpler way to derive and to understand abundance patterns.\n\nMaximum entropy can be thought of purely as a basic invariance method of analysis. Maximum entropy distributions have the form in Equation 1 as\n\n\n\nin which Tz is the affine-invariant scale that defines the probability pattern. Thus, the method of maximum entropy is simply a method for deriving the affine-invariant expression, Tz. In practice, maximum entropy has three limitations.\n\nFirst, maximum entropy is silent with respect to the proper choice for the scale on which entropy is maximized and the constraints that set the affine-invariant expression, Tz. By contrast, focus on invariance led us to the shift invariance of the process scale, r. That scale provided a much simpler analysis, in which r is the incremental scale with respect to invariance and the measurement scale with respect to entropy.\n\nIn other words, maximum entropy is a blind application of the most basic invariance principles, without any guidance about the proper scales for invariance, randomness, and constraint. By contrast, an explicit invariance approach takes advantage of the insight provided by the analysis of invariance.\n\nSecond, by focusing on invariance, we naturally obtain the full invariance (symmetry) group expression in Equation 3 as the generic form of probability patterns\n\n\n\nThat generic expression leads us to a generalization of the log series in Equation 5 as 24\n\n\n\nwhich is a two parameter distribution for abundances with respect to λ and β. The log series is a special case with β = 1.\n\nThird, invariance leads to a deeper understanding of the relation between observed pattern and alternative mechanistic models of process. The following section provides an example.\n\n\nNeutrality\n\nHere, we analyze Hubbell’s5 neutral model of species abundances in the light of our invariance perspective. With that example in mind, we then discuss more generally how neutral models relate to invariance and maximum entropy.\n\nThe strong recent interest in Hubbell’s neutral model follows from the match of the theory to the contrasting patterns of species abundance distributions (SADs) that have been observed at different spatial scales. In the theory, many local island-like communities are connected by migration into a broader metacommunity. Sufficiently large metacommunities follow the log series pattern of species abundances. Each local community follows a distribution that Hubbell called the zero-sum multinomial30, which is similar to a skewed lognormal. As noted by Rosindell et al.6, it is this flexibility of the classic neutral model to reconcile the log series and lognormal distributions that allows it to fit empirical data well31.\n\nBroad consensus suggests that species abundances closely follow the log series pattern at large spatial scales. Extensive data support that conclusion4.\n\nObserved pattern at small spatial scales differs from the log series. Consensus favors a skewed lognormal pattern. The data typically show an excess of rare species, causing a skew relative to the symmetry of the lognormal when plotted on a logarithmic scale.\n\nAt small spatial scales, most recent analyses focus on data from a single long-term study of tree species in Panama5,30. Thus, some ambiguity remains about the form and consistency of the actual pattern at small scales. The blue curve of Figure 3 shows Chisholm & Pacala’s30 fit of the neutral theory to the Panama tree data for species abundances at small spatial scales. The gold curve shows the close match to the neutral theory pattern by a simple probability distribution derived from the analysis of invariance.\n\nThe neutral theory fit to the data comes from Chisholm & Pacala’s30 analysis in their Figure 1. They used Hubbell’s neutral theory model with parameters J = 21,060, m = 0.075, and θ = 52.1 in their Equation 3, originally from Alonso & McKane32. The gamma-lognormal model in Equation 11 produces essentially the identical pattern with parameters λ = 0.00205, a = 0.491, and α = 0.0559. The abundance scale can be expressed equivalently on the process scale, log2n = r/ log 2. See the Zenodo record33 for the calculations used to produce this plot.\n\nTo obtain the matching distribution derived by invariance, we begin with the canonical form for probability distributions in Equation 3. That canonical form expresses pattern in terms of the shift-invariant scale, w. Next, we need to find the specific form of the scale w that relates this canonical form for probability distributions to the neutral theory. Because the neutral theory derives abundance, n, as an outcome of demographic processes, r, the fundamental shift-invariant scale for neutral theory is expressed in terms of the demographic process variable as\n\n\n\nBelow, we discuss why this is a natural shift-invariant scale for neutral theory. For now, we focus on the details of the mathematical expressions. Recall that n = er relates measured abundances, n, to the demographic process scale, r. If we assume that β = 1 in Equation 3 and use w from Equation 10, we obtain\n\n\n\nwith parameters λ, a, and α. We can write this distribution equivalently on the n scale for abundance as\n\n\n\nIn the second distribution, µ = (a – ã)/2α. Thus, both distributions have the same three parametric degrees of freedom.\n\nThe right-hand exponential term of Equation 12 is a lognormal distribution with parameters µ and σ2 = 1/2α. The remaining terms are a gamma distribution with parameters ã and λ. We call this product of the gamma and lognormal forms the gamma-lognormal distribution.\n\nFigure 3 showed that the gamma-lognormal distribution matches the neutral theory fit for the Panama tree data. Figure 4 shows that the shape of the gamma-lognormal matches the shape of the neutral theory predictions for various mechanistic parameters of the neutral theory.\n\nThe blue curve for the neutral theory and the gold curve for the gamma-lognormal are calculated as described in Figure 3. The parameters for the neutral theory are the same as in Figure 3, except as shown in each panel. We fit the parameters for the gamma-lognormal to each neutral theory curve, with values for each panel: (a) λ = 0.01115, a = 0.4452, and α = 0.03660; (b) λ = 0.0004209, a = 0.4622, and α = 0.05014; (c) λ = 0.002765, a = 0.3182, and α = 0; (d) λ = 0.001777, a = 0.2217, and α = 0.03576; (e) λ = 0.0001509, a = 0.3851, and α = 0.03667; (f) λ = 0.02519, a = 0.3726, and α = 0.006900. See the Zenodo record33 for the calculations used to produce these plots.\n\nIn summary, the neutral theory distribution appears to be nearly identical to a gamma-lognormal distribution when compared over realistic parameter values. Both distributions have the same three parametric degrees of freedom. Pueyo et al.25 derived the gamma-lognormal by using an invariance argument to obtain the n = er relation as a Bayesian prior for maximum entropy and then using additional constraints in a maximum entropy analysis. They also noted the good fit to Hubbell’s neutral theory. As mentioned above, our invariance analysis and our interpretation of invariance differ from Pueyo et al.’s Jayesian invariant prior approach for maximum entropy.\n\nThe constraints on pattern can be seen most clearly by rewriting Equation 11 as\n\n\n\nin which r˜2=(r−μ)2 is the squared deviation from µ, in which µ is the average value of r. This expression remains a three-parameter distribution because, as noted above, μ = (a–ã)/2α.\n\nWith this set of parameters, the affine-invariant scale is\n\n\n\nNote that T and w are related by Equation 2. We are using w from Equation 10 and β = 1, as noted below Equation 10. We ignore the extra –1 term in T of Equation 2, because the canonical form of probability distributions is invariant to adding a constant to T. The tilde parameters of the distribution in Equation 13 are interchangeable with the nontilde parameters of the identical distribution in Equation 12. The tilde expressions focus on the invariances that will help us to interpret ecological pattern. The nontilde expressions describe pattern in terms of the classic forms for the gamma and lognormal distributions.\n\nBy the standard theory of maximum entropy, qr maximizes entropy on the incremental scale dr subject to a constraint on the average value of the defining affine-invariant scale, ⟨T⟩r. That constraint is the linear combination of three constraints: the average abundance on the process scale, ⟨n = er⟩r, the average demographic process value, ⟨r⟩, and the variance in the demographic process values, 〈r˜2〉.\n\nBy maximum entropy, all of the information in Hubbell’s mechanistic process theory of neutrality and the matching gamma-lognormal pattern reduces to maximum randomness subject to these three constraints.\n\nHowever, it is very unlikely that we would have derived the correct form by maximum entropy without knowing the answer in advance. This limitation emphasizes that maximum entropy provides deep insight into process and pattern, but often we need an external theory to guide our choice among various possible maximum entropy formulations.\n\nPut another way, maximum entropy and process oriented theories, such as Hubbell’s model, often work together synergistically to provide deeper insight than either approach alone.\n\nBefore turning to invariance and the gamma-lognormal pattern of neutral theory, it is useful to consider some basic properties of invariance and information34,35. In particular, this subsection develops our claim that the affineinvariant scale provides the deepest insights into the relations between pattern and process.\n\nWe start by noting that, in the general expression for probability distributions\n\n\n\nthe affine-invariant scale, Tz, is equivalent to a common expression for the information content in a measurement, z, as\n\n\n\nThis expression follows from assuming that: information depends on the probability, qz, of observing the measured value and not on the value itself; rarely observed values provide more information than commonly observed values; and the information in two independent measurements is the sum of the information in each measurement. From the general expression for probability distributions\n\n\n\nThus, an incremental change in information is equal to an incremental change in the affine-invariant scale\n\n\n\nEquivalently, the change in information with respect to a change in the affine-invariant scale,\n\n\n\nis constant at all magnitudes of the measurement, z. Every measured increment on the Tz scale provides the same amount of information about pattern. Constancy of information at all magnitudes is the ideal for a measurement scale. Thus, affine-invariance provides the ideal scale on which to evaluate the pattern in measurements23. Figure 5 illustrates some key properties of the affineinvariant scale.\n\nA continuous distribution typically can be written as qz = ke–λTz, from Equation 1. In the figure, T ≡ Tz. (a) A parametric plot of qz vs Tz is exponential. All of differences between probability distributions are contained in the form of the affine-invariant scale, Tz. The change in information for each increment of the affine-invariant scale is λ, as in Equation 15. (b) A parametric plot of qz vs ±Tz, is normally distributed when describing the deviations from a unimodal peak of qz. The average of the deviations on the affine-invariant scale, ⟨T⟩, relative to measurements on the square root of that scale, Tz, is the variance, σ2. For the normal distribution, we can think of a deviation from the central location on the affine-invariant scale, Tz=Rz2, as the squared radial deviations along the circumference of a circle with radius Rz, describing the squared vector length for an aggregation of variables. The variance is the average of the squared radial deviations relative to the scale of radial measures, Tz=Rz. Most continuous unimodal distributions are, in this way, equivalent to a normal distribution when scaled with respect to the square root of the affine-invariant measure. See Frank17,18 for details.\n\nInformation is sometimes thought of as a primary concept. However, it is important to understand that, in this context, information and affine invariance are the same thing. Neither is intrinsically primary.\n\nWe prefer to emphasize invariance, because it is an explicit description of the properties that pattern and process must obey17,27,36. Further analysis of invariant properties leads to deeper insight. For example, only through invariance can we obtain the group theory expression for the canonical form of probability patterns (Equation 3).\n\nBy contrast, “information” is just a vague word that associates with underlying invariances. Further analysis of information requires unwinding the definitions to return to the basic invariances.\n\nWe turn now to the neutral theory model for abundances at local spatial scales. We showed that all of the information about pattern and process in the neutral theory is captured by the gamma-lognormal pattern in Equation 13 as\n\n\n\nwhich defines the affine-invariant scale in Equation 14 as\n\n\n\nOn this scale, changes in r provide the same amount of information about pattern at all magnitudes. Shifting the scale by a constant does not change the information about pattern in measurements. In other words, it does not matter where we set the zero point for Tr. Similarly, uniformly stretching or shrinking the scale, Tr, does not change the information in measurements of r.\n\nWe can parse the terms of Equation 16 with respect to constraint and invariance. When r is large, the term λer = λn dominates the shape of the distribution in the upper tail, which decays as\n\n\n\nfor sufficiently large er = n. The smaller the value of λ relative to ã and α, the greater er must be for this pattern to dominate. When λ is relatively large compared with ã and α, this pattern dominates at all magnitudes and leads to the log series.\n\nWith respect to constraint, for large values of abundance, n, the constraint on average abundances dominates the way in which altered process influences pattern. With respect to invariance, a process that additively shifts or multiplicatively stretches the er = n values does not alter the pattern in the upper tail. Similarly, pattern is invariant to a process that additively shifts process values, r, but processes that multiplicatively change r alter pattern. Thus, we can evaluate the role of particular processes by considering how they change n or r.\n\nThe pattern at small and intermediate values of r depends on the relative sizes of the parameters. If the ãr term dominates, then the constraint, ⟨r⟩, on the average process value is most important. With respect to invariance when ãr dominates, a process that additively shifts or multiplicatively stretches the r values does not alter the pattern in the lower tail. That lower tail is a rising exponential shape, eãr, as in Figure 4c.\n\nWhen the αr˜2 term is negligible at all magnitudes, the combination of the dominance by ãr in the lower tail, and the dominance by λer in the upper tail, yields the gamma distribution pattern on the abundance scale, n.\n\nFinally, for magnitudes of r at which the αr˜2=α(r−μ)2 term dominates, the constraint, σ2 = ⟨r – µ2⟩, on the variance in process values is most important. In this case, pattern follows a normal distribution, e–α(r–µ)2, on the r scale, which is a lognormal distribution on the abundance scale, n.\n\nWhen combining numerous process values to obtain an overall net r value, approximate rotational invariance is sufficient for the pattern to be very close to a perfect normal curve (see Introduction). When measuring net squared deviations from the mean, which is the squared radial distance, the pattern is invariant to shift and stretch of the squared radial measures, (r – µ)2.\n\nIn practice, the lognormal pattern of abundance dominates when a constraint on r dominates and net values of r obey rotational invariance (symmetry) with respect to the summing up of the individual processes acting on abundance.\n\nAny theory of process that leads to those three basic invariances will follow the gamma-lognormal pattern. The great unsolved puzzle is how specific mechanistic processes combine such that the structure of pattern is fully expressed by these particular invariances of pattern or, equivalently, by constraints on the average values of certain quantities in the context of maximum entropy. Our work opens the way for a more direct attack on this great puzzle by clarifying the anatomy of a pattern, thereby clarifying the puzzle that must be solved.\n\n\nThe anatomy of pattern\n\n[J]ust as the physiologist divides the animal world, according to anatomy, into families and classes, so the ornamentist is able to classify all pattern-work according to its structure [invariance]. Like the scientist, he is able even to show the affinity between groups to all appearance dissimilar; and, indeed, to point out how few are the varieties of skeleton upon which all this variety of effect is framed (ref. 37, pp. 3–4). … The fact of the matter is, the characteristic lines of time-honoured patterns are mainly the direct result of the restrictions under which the craftsman was working (ref. 37, p. 47).\n\nInvariances comprise the structural components in the anatomy of pattern. Commonly observed patterns almost always dissect completely into a few simple invariances. Our primary goal has been to introduce into ecological study the anatomy of pattern and the methods of dissection.\n\nIdentifying and naming the parts does not tell one how those parts came to be. In fact, common patterns are widespread exactly because so many different underlying mechanistic processes give rise to the same simple invariances.\n\nRoughly speaking, one can think of a common pattern as an attractor. Each different underlying mechanistic process that develops into the generic form traces a distinctive path from some starting point to the generic endpoint of the attractor. All of the different mechanistic processes and starting points that end up at the same attractor form the basin of attraction for that pattern.\n\nOur work characterized the anatomy of pattern—the anatomy of the attractors. The next step requires understanding how various combinations of mechanistic processes lead to one attractor or another. Equivalently, one can think of a mechanistic process as something that transforms inputs into outputs38. Three questions follow. How do particular cascades of input-output transformations ultimately combine to produce overall transformations that associate with simple invariances? What separates some cascades from others with regard to association with different invariances? In other words, how can we assign different mechanistic cascades to one basin of attraction or another?\n\nIf we could answer those questions, then we could predict whether different mechanistic processes lead to the same pattern or to different patterns.\n\nThe fact that different processes can attract to the same pattern has been widely discussed in ecology30,39–47. However, that past work typically did not explain common patterns in terms of invariance. Without invariance, one does not have a basis for describing the anatomy of common patterns or the reasons why certain processes attract to a particular pattern and others do not.\n\nInvariance may provide a way to compare different models of process that lead to the same pattern. Among the many complex component processes that may occur in a model, which truly matter? In other words, which component processes shape the defining invariances and which are irrelevant? For the focal component processes of each model that matter, which empirical tests would tell us which of the alternative mechanistic models is the more likely match to natural processes?\n\n\nConclusions\n\nThe apparent simplicity of invariance can mislead about its ultimate power. For example, probability patterns express a shift and stretch invariant scaling. That affine-invariant scaling provides a constant measure of information at all magnitudes.\n\nShift and stretch invariance seem almost trivially simple. Yet, by analyzing how repeated transformations of shift and stretch retain invariance, we obtain the most general form that expresses various affine-invariant scales (Equation 2). That affine symmetry group defines the simple, general structure of probability patterns and their uniform measurement scales.\n\nKnowing the general invariant form of probability patterns reveals the relations between different approaches. Invariance provides powerful methods to analyze pattern and process.\n\nTo sum up, our invariance approach is not just another one among various alternatives. Rather, it is the only way to relate process to pattern, because the essence of pattern is invariance. Only by understanding what pattern actually is and how it generally arises can one begin to formulate testable hypotheses about mechanism.\n\nPut another way, pattern is always the interaction between, on the one hand, the generic aspects of invariance and scale that arise in all cases and, on the other hand, the particular aspects of biology that operate in each case. Without a clear view of that duality between the generic and the particular, it is easy to mistakenly attribute generic aspects of observed pattern to particular causes. To properly understand the role of specific mechanistic aspects in shaping pattern, one must evaluate pattern simultaneously from the perspectives of the generic and the particular.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nSoftware availability\n\nThe Mathematica code for the analysis and creation of Figure 3 and Figure 4 is available at Zenodo: https://doi.org/10.5281/zenodo.324336433.\n\nLicense: Creative Commons Attribution 4.0 International license.", "appendix": "Author contributions\n\n\n\nSAF initiated the project, did the mathematical analyses, created the figures, and wrote the first draft. JB developed connections to broader issues in ecology, expanded the text to clarify exposition and significance, extended the framing of concepts and the explanation and discussion of key points, and edited the entire manuscript.\n\n\nAcknowledgments\n\nSAF completed this work while on sabbatical in the Theoretical Biology group of the Institute for Integrative Biology at Eidgenössische Technische Hochschule (ETH) Zürich.\n\nPrevious versions of this article are available on bioRxiv: https://doi.org/10.1101/673590.\n\n\nReferences\n\nLederman LM, Hill CT: Symmetry and the Beautiful Universe. Prometheus Books, Amherst, N.Y. 2004. Reference Source\n\nFisher RA, Corbet AS, Williams CB: The relation between the number of species and the number of individuals in a random sample of an animal population. J Anim Ecol. 1943; 12(1): 42–58. Publisher Full Text\n\nPreston FW: The commonness, and rarity, of species. Ecology. 1948; 29(3): 254–283. Publisher Full Text\n\nBaldridge E, Harris DJ, Xiao X, et al.: An extensive comparison of species-abundance distribution models. PeerJ. 2016; 4: e2823. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHubbell SP: The Unified Neutral Theory of Biodiversity and Biogeography. Princeton University Press, Princeton, NJ, 2001. Reference Source\n\nRosindell J, Hubbell SP, Etienne RS: The unified neutral theory of biodiversity and biogeography at age ten. Trends Ecol Evol. 2011; 26(7): 340–8. PubMed Abstract \| Publisher Full Text\n\nBrown JH: Macroecology. University of Chicago Press, Chicado, 1995. Reference Source\n\nGaston KJ, Blackburn TM: Pattern and Process in Macroecology. Blackwell Science, Oxford, UK. 2000. Publisher Full Text\n\nHarte J: Maximum Entropy and Ecology: A Theory of Abundance, Distribution, and Energetics. Oxford University Press, New York, 2011. Publisher Full Text\n\nJaynes ET: Information theory and statistical mechanics. Phys Rev. 1957; 106(4): 620–630. Publisher Full Text\n\nJaynes ET: Information theory and statistical mechanics. II. Phys Rev. 1957; 108(2): 171–190. Publisher Full Text\n\nJaynes ET: Probability Theory: The Logic of Science. Cambridge University Press, New York. 2003. Publisher Full Text\n\nWagensberg J, Valls J: The extended maximum entropy formalism and the statistical structure of ecosystems. Bull Math Biol. 1987; 49(5): 531–538. Publisher Full Text\n\nWagensberg J, Lopez D, Valls J: Statistical aspects of biological organization. J Phys Chem Solids. 1988; 49(6): 695–700. Publisher Full Text\n\nSherwin WB, Fornells NP: The introduction of entropy and information methods to ecology by Ramon Margalef. Entropy. 2019; 21(8): 794. Publisher Full Text\n\nAlonso D, Ostling A, Etienne RS: The implicit assumption of symmetry and the species abundance distribution. Ecol Lett. 2008; 11(2): 93–105. PubMed Abstract \| Publisher Full Text\n\nFrank SA: The invariances of power law size distributions. F1000Res. 2016; 5: 2074. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFrank SA: Common probability patterns arise from simple invariances. Entropy. 2016; 18(5): 192. Publisher Full Text\n\nSolé RV, Manrubia SC, Benton M, et al.: Criticality and scaling in evolutionary ecology. Trends Ecol Evol. 1999; 14(4): 156–160. PubMed Abstract \| Publisher Full Text\n\nJordano P, Bascompte J, Olesen JM: Invariant properties in coevolutionary networks of plant-animal interactions. Ecol Lett. 2003; 6(1): 69–81. Publisher Full Text\n\nStouffer DB, Camacho J, Guimerà R, et al.: Quantitative patterns in the structure of model and empirical food webs. Ecology. 2005; 86(5): 1301–1311. Publisher Full Text\n\nFrank SA, Smith E: A simple derivation and classification of common probability distributions based on information symmetry and measurement scale. J Evol Biol. 2011; 24(3): 469–484. PubMed Abstract \| Publisher Full Text\n\nFrank SA: How to read probability distributions as statements about process. Entropy. 2014; 16: 6059–6098. Publisher Full Text\n\nFrank SA: The common patterns of abundance: the log series and Zipf's law. F1000Res. 2019; 8: 334. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPueyo S, He F, Zillio T: The maximum entropy formalism and the idiosyncratic theory of biodiversity. Ecol Lett. 2007; 10(11): 1017–1028. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarte J, Zillio T, Conlisk E, et al.: Maximum entropy and the state-variable approach to macroecology. Ecology. 2008; 89(10): 2700–2711. PubMed Abstract \| Publisher Full Text\n\nFrank SA: Invariant death. F1000Res. 2016; 5: 2076. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFrank SA: Measurement scale in maximum entropy models of species abundance. J Evol Biol. 2011; 24(3): 485–496. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPueyo S: Comparison of two views of maximum entropy in biodiversity: Frank (2011) and Pueyo et al. (2007). Ecol Evol. 2012; 2(5): 988–993. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChisholm RA, Pacala SW: Niche and neutral models predict asymptotically equivalent species abundance distributions in high-diversity ecological communities. Proc Natl Acad Sci U S A. 2010; 107(36): 15821–15825. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEtienne RS, Rosindell J: The spatial limitations of current neutral models of biodiversity. PLoS One. 2011; 6(3): e14717. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlonso D, McKane AJ: Sampling Hubbell’s neutral theory of biodiversity. Ecol Lett. 2004; 7(10): 901–910. Publisher Full Text\n\nFrank SA: Mathematica code for \"Invariance in ecological pattern\". 2019. http://www.doi.org/10.5281/zenodo.3243365\n\nTribus M: Thermostatics and thermodynamics an introduction to energy, information and states of matter, with engineering applications. Van Nostrand, New York, 1961. Reference Source\n\nCover TM, Thomas JA: Elements of Information Theory. Wiley, New York, 1991. Reference Source\n\nFrank SA: Measurement invariance explains the universal law of generalization for psychological perception. Proc Natl Acad Sci U S A. 2018; 115(39): 9803–9806. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDay LF: The Anatomy of Pattern. B. T. Batsford, London, 1887. Reference Source\n\nFrank SA: Input-output relations in biological systems: measurement, information and the Hill equation. Biol Direct. 2013; 8: 31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCohen JE: Alternate Derivations of a Species-Abundance Relation. Am Nat. 1968; 102(924): 165–172. Publisher Full Text\n\nChave J, Alonso D, Etienne RS: Comparing models of species abundance. Nature. 2006; 441: E1–E2. PubMed Abstract \| Publisher Full Text\n\nAdler PB, Hillerislambers J, Levine JM: A niche for neutrality. Ecol Lett. 2007; 10(2): 95–104. PubMed Abstract \| Publisher Full Text\n\nZillo T, Condit R: The impact of neutrality, niche differentiation and species input on diversity and abundance distributions. Oikos. 2007; 116(6): 931–940. Publisher Full Text\n\nEtienne RS, Alonso D, McKane AJ: The zero-sum assumption in neutral biodiversity theory. J Theor Biol. 2007; 248(3): 522–536. PubMed Abstract \| Publisher Full Text\n\nFrank SA: The common patterns of nature. J Evol Biol. 2009; 22(8): 1563–1585. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcGill BJ: Towards a unification of unified theories of biodiversity. Ecol Lett. 2010; 13(5): 627–642. PubMed Abstract \| Publisher Full Text\n\nMatthews TJ, Whittaker RJ: Neutral theory and the species abundance distribution: recent developments and prospects for unifying niche and neutral perspectives. Ecol Evol. 2014; 4(11): 2263–2277. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMatthews TJ, Whittaker RJ: Fitting and comparing competing models of the species abundance distribution: assessment and prospect. Front Biogeogr. 2014; 6(2): 2. Publisher Full Text" }	[ { "id": "58055", "date": "24 Jan 2020", "name": "David Alonso", "expertise": [ "Reviewer Expertise community ecology", "population biology", "infectious diseases", "biodiversity research", "climate change", "environmental forcing", "stochastic birth-death processes", "non-linear interactions", "self-organization", "complex systems" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis contribution emphasizes how the concept of invariance connects patterns to processes leading to an original synthesis of previous approaches. In particular, this piece of work illustrates the application of the concept of invariance to deduce the most commonly observed species abundance distributions (SAD) in nature. The authors succeed in giving a general overview of the relation between invariance, process, and pattern.\nAs an illustration, they present two key theoretical results. First, they derive the log series SAD from a simple maximum entropy argument. When species abundances fluctuate randomly on a log scale, this is, process values 'r' are random (where r is defined as log n), and these fluctuations are only subject to an ecological constraint limiting total abundance, then the log series distribution, initially introduced by R. A. Fisher, naturally arises. Second, they show that Hubbell's neutral model is the simple expression of three basic invariances, which correspond to a maximum entropy model constraining average species abundances, and the average and variance of the demographic processes influencing abundance.\n\nThe first theoretical result is far from original as the same argument is clearly presented by Pueyo et al.(2007)1 in \"The maximum entropy formalism and the idiosyncratic theory of biodiversity\" where clearly, after a scale invariance argument is done, the log series again naturally arises from a maximum entropy derivation by only constraining on total abundance. However, the second result and the whole emphasis on the generality and the power of the invariance approach in ecology is a true novel contribution to the field.\n\nThe authors state, in their conclusions, that \"the invariance approach is the only way to relate process to pattern\". The authors emphasize that, in order to uncover the plausible underlying mechanisms underlying an observed pattern, one needs first to pay special attention to the general invariant form of probability patterns. In the future, I would like to see the authors' invariance approach to apply, more generally to other patterns in ecology.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5254", "date": "24 Feb 2020", "name": "Steven Frank", "role": "Author Response F1000Research Advisory Board Member", "response": "We thank David Alonso for his thoughtful comments about our article. We agree that trying to apply the invariance approach to additional ecological patterns remains an important challenge for future work." } ] }, { "id": "57848", "date": "07 Feb 2020", "name": "Neil McRoberts", "expertise": [ "Reviewer Expertise Epidemiology", "decision theory", "information theory", "quantitative biology", "policy analysis." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper by Frank and Bascompte explores ideas about the origins of statistical pattern in biology developed in previous work by Frank and Frank & Smith, and applies them to the question of the distributions of species population sizes commonly observed in community ecology. As the authors note, the distribution of population sizes in a given ecological community will tend to follow quite closely one of two probability distributions – the logarithmic or the skewed log-normal – irrespective of the physical size of the organisms in the community of interest, and the number of species involved. Why this should be is an important question, and one that points toward some general rule or principle of ecology that would be fascinating and potentially valuable to understand. In providing their own explanation, the authors follow a formula proven by Frank in earlier publications: The problem is outlined, previous efforts to explain it are described, noting ad hoc or special features which limit their generality, before the authors’ own explanation is described and its relative merits compared with the earlier attempts.\nIt is worth taking a moment to put this work in a brief historical context of the more general topic of research that has occupied Frank for more than a decade, because that broader (longer?) perspective says something useful about the process of research into theoretical biology and the development of theory. Frank’s earliest publications on the topic of probability patterns in nature were built around the idea that constraints on information contained in myriad stochastic processes, together with a principle of maximum entropy, leads to a few probability patterns being common. This explanation raises the question of how the constraints occur. Frank’s earlier work led to some principles to answer that question by assuming that the constraints arise in solving the maximum entropy expression for probability patterns with the method of Langrangian multipliers. Of course, since what’s needed is an explanation for empirical observation, none of this is supposed to be happening to purely abstract mathematical objects, but to actual physical processes, when we observe them (and indeed some of the most important constraints on information in our data arise from how we observe), so an explanation in terms of constraints in Lagrangians had better have a clear and fairly direct physical interpretation; fortunately it does.\nA natural interpretation of the Lagrangian constraints arises in thinking about the scaling at which information is gathered from the system by the process of observation and how this interacts with natural scales at which information is preserved in the organization of the observed system. Earlier publications, rooted in the maximum entropy concept, used the idea of convolution to describe how information could be preserved (or lost) as large numbers of random, small scale, processes interact and are observed. This line of thought appears to have led Frank to the realization that the organizing framework for the constraints is, at its core, a set of expressions describing patterns of invariance. Three types of invariance – shift and stretch (which combine to give affine invariance) and rotation, are sufficient to account for the fact that a few common probability patterns in biological and physical systems spanning many orders of magnitude, describe the majority of empirical datasets. Frank & Smith (2011)1 gives a comprehensive early account of using the concept of symmetries (invariances) to derive many probability patterns. A recent strand of papers (of which the current one is an example) employ the concept of invariance to show how several specific types of biological pattern arise.\nIn these papers, the concept of invariance is given primacy, and - explicitly in the current example - maximum entropy is viewed as a derived property that is not needed to explain the commonality of probability patterns. To reiterate an earlier point, quite aside from the technical merits, theoretical depth, and potential applications of the work itself, Frank’s publications on this topic are an interesting publication trail for those studying the development of theory in biology. They present an example of how one scientist’s thinking on a subject changes and develops over time. I’m laboring the description of the context for the work, because, as I will outline in what follows, I think the most important critique lies not in the technical details as they apply to species abundance distributions, but to the epistemic basis for the whole endeavor.\nInvariance and maximum entropy Frank & Bascompte (F&B) argue that the log series for species abundance arises naturally when one considers two assumptions; first that there is affine invariance at the scale of proportional processes that act on species abundance (so, for example, birth and death), and second that there is a constraint on average abundance. Algebraic analysis, assuming a standard exponential form for the probability distribution, then shows that these two assumptions are sufficient to induce the log-series form; the probability distribution describing abundance. The authors contrast this analysis with the one owing to Harte, which is based on a maximum entropy interpretation. F&B note that working from first principles, assuming a constraint on average abundance, and starting from the assumption that entropy is maximized in the abundance distribution, one ends up with the exponential distribution as the maximum entropy form for species abundance. This is at odds with empirical observation.\nIf we assume that the observed distribution of species abundances is a maximum entropy distribution, then this analysis tells us that simple constraint on the average abundance is insufficient to induce the observed probability pattern, which leads to three possible alternatives. First, some further constraint is required on the abundance distribution so that the derived form matches observation (this is essentially the approach taken by Harte). Second, the abundance distribution is itself dependent on one or more constraints in some other process (for which entropy is maximized) and the joint effect of these two sets of constraints results in the observed log series distribution (this is the approach taken by F&B). Thirdly, we abandon the premise that species abundance is a maximum entropy distribution and look for explanations in some other room in the library of all possible theories. The third option is a drastic one; especially when there are good arguments in related fields of research that support the idea that Nature does indeed confront us with maximum entropy distributions when we make observations. For example, in discussing the correspondence between entropy maximization and description length minimization, Grunwald (2007, p644)2 writes:\n“we imagine a two-player game between Nature and Statistician. Nature first picks a distribution and then generates an outcome X according to it; Statistician picks a code and uses it to describe X. Nature is allowed to select any distribution she likes, as long as it satisfies E[ϕ(X)]=μ, and Statistician is allowed to use any code whatsoever. Nature’s goal is to maximize Statistician’s expected codelength, and Statistician’s goal is to minimize it. … the best (maximum codelength_ that she can achieve if she has to move first is equal to the best (minimum codelength) that Statistician can achieve if he has to move first. Surprisingly, under weak conditions both Nature’s maximin and Statistician’s minimax strategy turn out to be the Maxent distribution…”\nThere is, I believe, an important connection between MDL and Frank’s program of explanation for biological patterns and it is captured in the quotation from Grunwald’s (2007)2 book given above. In a loose sense, one might cast Frank’s investigation of pattern as an inquiry into what Nature is doing in the game described by Grunwald. The conclusion is that she is playing a strategy of showing us maximum entropy distributions. As Grunwald (2007)2 points out, this is the optimal conclusion for us to reach (playing the role of Statistician) if we want our adopted descriptions to be optimal in the sense of minimizing our expected maximum error. The Kraft-McMillan inequality establishes the correspondence between codelength functions and probability distributions, so Grunwald’s game between Nature and Statistician can be rephrased directly by substituting “probability distribution” for “codelength”. But, there is an additional, epistemic, connection between MDL and what Frank and his co-authors on this and other papers are doing. In establishing MDL Jorma Rissanen was attempting to establish a principle for model selection and inference that was free from the need for prior assumptions about the process (or model) generating the observed data. Here is the opening paragraph of Rissanen’s (1978)3 paper on model selection:\nThis study is an attempt to derive a criterion for estimation of both the integer-valued structure parameters and the real-valued parameters of dynamic systems starting from a single natural and fundamental principle: the least number of digits it takes to write down an observed sample of a time series.\nFrank’s scheme, for describing why particular models (i.e. probability distributions) describe is an alternative, but also hypothesis-free, attempt to describe what Nature is doing. It’s important to clarify what is meant in saying Frank’s approach is hypothesis-free. It is simply this. The method does not select a particular probability distribution (equivalently, a model or codelength function) for the data a priori, but instead establishes a few mechanistically motivated constraints on information, given the context of the data and the measuring process, and uses those to infer the form of the probability distribution one expects the data to follow. This idea of making well-motivated choices about the identity of the best description of observed data is also enshrined in MDL in the “luckiness principle” (see Grunwald (2007)2 Ch14) further emphasizing the connection between the two lines of investigation.\nWhy does this matter in relation to the paper by F&B? As I mentioned earlier the answer is more one of process and principle than technical detail. The importance of the current paper is that it adds to argument, advanced by Frank, that biological observations of all kinds can be systematized; general principles operate that allow us to form expectations about the distributional properties of our data in a non ad hoc manner. I applaud this effort and think that it’s a contribution to modern biology that will come to be seen as a major advance in the philosophical grounding of the subject, which is why I find the current paper somewhat frustrating. My main concern is this. In seeking to establish an invariance principle as taking precedence in some epistemiological sense, over the principle of maximum entropy, I think the authors make a mistake, and one that threatens the clarity of the preceding work by Frank and others on probability patterns. In essence the problem is that invariance and maximum entropy are not alternatives, the former is one of two approches commonly used to solve/understand maximum entropy problems, the other being the method of Lagrangian multipliers. I would characterize the conceptual shift in this paper (from Frank’s previous work) not as a shift from maximum entropy to invariance, but as a shift in focus on solutions to the maxent problems from approaches grounded in Lagrangians to approaches derived from the concepts of invariances, particularly symmetry groups. Both approaches are firmly within the overall framework of maximum entropy. So, my main request to the authors would be that they consider re-casting the paper along the lines just outlined and less as a demonstration that a principle of invariance supersedes the maximum entropy principle in describing biological patterns, in particular species abundance distributions. To anchor this argument more firmly to the paper (and draw in the MDL connection) here are a couple of points where I think the authors need to offer the reader a little more support for their proposal. F&B argue that their approach, based on invariance, offers a clearer rationale for deriving/explaining appropriate distributional forms than those based in either “maximum entropy” or mechanistic neutral theories. For example, in the section “Maximum entropy and the gamma lognormal” F&B note:\nBy maximum entropy, all of the information in Hubbell’s mechanistic process theory of neutrality and the matching gamma-lognormal pattern reduces to maximum randomness subject to these three constraints.\nHowever it is very unlikely that we would have derived the correct form by maximum entropy without knowing the answer in advance. This limitation emphasizes that maximum entropy provides deep insight into process and pattern, but often we need an external theory to guide our choice among various possible maximum entropy formulations. I would argue that subsequent derivation based on invariances is no less opaque, and someone attempting the derivation would, similarly, need to know where they were going in order to get there. It is of interest to point out that here, as in MDL, access to external theory will be of value in achieving results. This idea of externally motivated choice of approach is also apparent in the second example I would ask the authors to consider. In the discussion of the log series pattern, F&B contrast their approach, with that offered by Harte. As we already noted, F&B point out that starting from the canonical form for probability distributions, a constraint on the expected value for the observations leads to the exponential distribution as the emergent form; a result that demonstrably fails to deal with observed species abundance data. Harte solved this problem by introducing a second variable that is similarly constrained at the same scale as the expectation of abundance. F&B criticize Harte’s approach, in essence, on the grounds that it is an ad hoc solution, arguing that their approach, in which constraints (invariances) are placed on underlying demographic processes, has a clearer rationale. While F&B’s argument is persuasive, I think it needs to be strengthened and somewhat expanded. Here’s why:\nAs a I pointed out above, failure of the simple constraint on average abundance to lead to the log series requires us to come up with an alternative hypothesis for why the log series is observed. In MDL terms, we need a better description of the data. From a logical perspective, there doesn’t appear to be any difference between adding an assumption of a variable at the same scale as abundance being under constraint, and an assumption of proportional demographic processes at a lower scale being constrained. In fact, from a model parsimony (MDL) perspective, a model that relies on adding a whole additional scale of processes may be viewed as propagating unnecessary complication. Furthermore, it doesn’t seem like too much of a stretch to suggest that constraints on proportional processes at a lower scale, might not give rise to quantity at the same scale as the expectation of abundance that is similarly constrained, when measured at that scale? Is the issue simply one of how phenomenological one likes one’s models to be? And if so, what guidance can be given to those who want to pursue the type of analysis proposed by F&B? As a first stab at an answer to that question would something along the following lines offer budding invariance analysts a template to work from?\nIdentify the Objects that are the subject of your interest (SAD’s in the current case). Use Occam’s Razor and Methodological Individualism, when deciding where to look for constraints. (IOUORMI “I owe you, or me”). The combination of Occam’s Razor and Methodological Individualism should guide investigators to look for the simplest model built from processes operating at one level below the objects of interest in the organizational hierarchy of the systems of interest.\nIn any case, I applaud the authors on showing that the diversity of known SAD data can be organized and explained by a unified principle, that has a clear theoretical and physical basis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5255", "date": "24 Feb 2020", "name": "Steven Frank", "role": "Author Response F1000Research Advisory Board Member", "response": "We are grateful for Neil McRoberts' careful reading and deep scholarship. We learned a lot. We agree with many points. Here, we confine our comments to a few issues that deserve further study. 1. \"My main concern is this. In seeking to establish an invariance principle as taking precedence in some epistemological sense, over the principle of maximum entropy, I think the authors make a mistake, and one that threatens the clarity of the preceding work by Frank and others on probability patterns. In essence the problem is that invariance and maximum entropy are not alternatives, the former is one of two approaches commonly used to solve/understand maximum entropy problems, the other being the method of Lagrangian multipliers. I would characterize the conceptual shift in this paper (from Frank’s previous work) not as a shift from maximum entropy to invariance, but as a shift in focus on solutions to the maxent problems from approaches grounded in Lagrangians to approaches derived from the concepts of invariances, particularly symmetry groups. Both approaches are firmly within the overall framework of maximum entropy.\" In the general form for probability density functions, q(z)=ke-λ T(z), we must distinguish two questions. First, how do we determine T(z)? That is the problem discussed by McRoberts in his comment quoted above. It is true that in our work we have, over time, moved from constraints and Lagrangians to symmetry groups in thinking about the form of T. Second, how does the overall exponential form of probability patterns arise? In our early work, we followed Jaynes in his maximum entropy approach to arrive at the exponential form. However, in our later work (e.g., ref 18), we realized that maximum entropy is itself a special form that arises within a more general understanding of invariance. From this more general perspective, \"entropy\" and \"information\" are simply expressions for basic invariances that one naturally assumes for probability patterns. There is no need for \"entropy\" as a concept or method, although there can be value in using notions of entropy in some applications and for developing intuitive understanding. For these reasons, we disagree with McRoberts' description of our work. We did intend to say that we have shifted from maximum entropy to a more general invariance perspective. From that more general perspective, one sees maximum entropy as a special interpretation of broader invariance principles. 2. \"[I]t is very unlikely that we would have derived the correct form [for Hubbell's neutral theory] by maximum entropy without knowing the answer in advance. This limitation emphasizes that maximum entropy provides deep insight into process and pattern, but often we need an external theory to guide our choice among various possible maximum entropy formulations. I would argue that subsequent derivation based on invariances is no less opaque, and someone attempting the derivation would, similarly, need to know where they were going in order to get there.\" We clearly pointed out in the article the dependence of our specific formulation on Hubbell's prior work. However, we also emphasized in our section \"The anatomy of pattern\" that there is room for new work that would get us out of the current limitations to understanding. That section discusses how one might connect, at a fundamental level, mechanistic models that generate pattern to particular invariances. If one could learn the general approach for connecting mechanism to invariance, then one may achieve a significant advance in insight. One may also find a method for developing testable hypotheses to differentiate between different causal mechanisms. 3. McRoberts argues that Harte's choice of an energy constraint and our choice of a scaling for growth processes are just two alternative ways to fit the data. We disagree. Similar probability patterns are very commonly observed across many disciplines for which an energy constraint does not make sense (see ref 24). Our choice for scaling arises from growth processes. Growth scaling is simply the process of multiplication, which is about as close to a truly fundamental concept as one can achieve. Although we did contrast our approach with Harte's work, we do not intend criticism of his approach. Instead, we see the important contributions to maximum entropy by Harte and others as the foundation on which we built our own novel approach. Great work naturally leads to further attempts to extend and to generalize, along with a continuing and lively conversation. For a topic as important as the interpretation of pattern, no one gets the last word. These few points which remain open for discussion highlight how much more there is to learn about the study of pattern. The thoughtful commentary provided by McRoberts is just the sort of thing that we need to challenge current understanding and help to push the field ahead. Thank you." } ] } ]	1	https://f1000research.com/articles/8-2093
https://f1000research.com/articles/8-1042/v1	10 Jul 19	{ "type": "Method Article", "title": "Development of an IgY-based lateral flow immunoassay for detection of fumonisin B in maize", "authors": [ "Tien Viet Tran", "Binh Nhu Do", "Thao Phuong Thi Nguyen", "Tung Thanh Tran", "Son Cao Tran", "Ba Van Nguyen", "Chuyen Van Nguyen", "Hoa Quang Le", "Tien Viet Tran", "Binh Nhu Do", "Thao Phuong Thi Nguyen", "Tung Thanh Tran", "Son Cao Tran", "Ba Van Nguyen", "Chuyen Van Nguyen" ], "abstract": "Fumonisin is one of the most prevalent mycotoxins in maize, causing substantial economic losses and potential health risks in human and animals. In the present study, in-house polyclonal IgY antibody against fumonisin group B (FB) was applied for the development of a competitive lateral flow immunoassay detecting these mycotoxins in maize grains with the limit of detection of 4000 µg/kg, which corresponds to the maximum residue limit adopted by The International Codex Alimentarius Commission. To this end, factors affecting the test performance including nitrocellulose membrane type, dilution factor of maize homogenates in running buffer, amount of detection conjugate, and incubation time between detection conjugate and samples were optimized. Under the optimal condition (UniSart® CN140 nitrocellulose membrane, FB1-BSA immobilized at 1 µg/cm, 1:10 dilution factor, 436 ng of gold nanoparticle conjugate, 30 minutes of incubation), the developed test could detect both FB1 and FB2 in maize with limit of detection of 4000 µg/kg, and showed no cross-reactivity to deoxynivalenol, ochratoxin A, aflatoxin B1 and zearalenone. When applied to detect FB1 and FB2 in naturally contaminated maize samples, results obtained from the developed assay were in good agreement with those from the high-performance liquid chromatography method. This lateral flow immunoassay is particularly suitable for screening of fumonisins in maize because of its simplicity and cost-effectiveness.", "keywords": [ "fumonisin B", "rapid methods", "lateral flow immunoassay", "IgY" ], "content": "Introduction\n\nFumonisins are a group of mycotoxins from Fusarium species, mostly Fusarium proliferatum and Fusarium verticillioides (Gelderblom et al., 1988). In term of chemical and physical characteristics, fumonisins are soluble in water and methanol; and are heat-stable over a wide range of processing unless the temperature exceeds 150°C (Jackson et al., 1996; NTP, 2001; Yazar & Omurtag, 2008). To date, four groups of fumonisin have been identified (A, B, C and P-series) (Rheeder et al., 2002), among which fumonisin B (FB) is the most common mycotoxins found in corn, and have been shown to have various toxic and carcinogenic effects (Munkvold et al., 2019; NTP, 2001). Due to its potential toxicity, the International Codex Alimentarius Commission has adopted the maximum level for the presence of fumonisins in raw maize at 4000 µg/kg (EC, 2005).\n\nSeveral recent studies pointed out fumonisin contamination in corn represents a major public-health concern in diverse countries including China (Fu et al., 2015; Guo et al., 2016; Hu et al., 2019; Liu et al., 2017), Brazil (Scussel et al., 2014), Kenya (Mutiga et al., 2015), South Africa (Mngqawa et al., 2016), Malawi (Mwalwayo & Thole, 2016), Tanzania (Kamala et al., 2016), Nigeria (Chilaka et al., 2016), Ethiopia (Getachew et al., 2018), Somalia (Wielogorska et al., 2019). In Vietnam, Hieu Phuong et al. (2015) showed that FBs were the major mycotoxin that contaminated maize with 67% of incidence, a range of positive samples for FB1 and FB2 at 102 to 10799 µg/kg and 102–5051 µg/kg respectively.\n\nConventionally, FBs could be detected by chromatography and immunological methods. To date, high performance liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) methods have been developed for FBs analysis. However, they are laborious, time consuming and require specialized equipment. On the other hand, lateral flow immunoassays (LFIA) are cost-effective, easy to use and suitable for on-site analysis. Several LFIAs have been developed for sensitive detection of FBs using monoclonal antibodies (Wang et al., 2013; Wang et al., 2014; Yu et al., 2015) or rabbit polyclonal antibodies (Anfossi et al., 2010; Venkataramana et al., 2014).\n\nPolyclonal IgY antibodies from egg yolk of laying hens represent an attractive alternative to monoclonal and rodent polyclonal antibodies. With one course of immunization, IgY could be extracted non-invasively in a large quantity (up to 40–80 mg), with 2–10% of which being antigen specific (Kovacs-Nolan & Mine, 2004; Pauly et al., 2011). As a result, IgY has been increasingly employed for the development of cost-effective rapid tests. Its usefulness in LFIAs has been demonstrated for detection of morphine (Gandhi et al., 2009), methicillin-resistant Staphylococcus aureus (Yamada et al., 2013), staphylococcal enterotoxins (Jin et al., 2013), and rhein (Zhang et al., 2018).\n\nIn the present study, we demonstrated the development of a gold nanoparticle-based LFIA that used in-house polyclonal IgY for simple and cost-effective screening of FBs in maize.\n\n\nMethods\n\nConjugation of FB1 to BSA was performed following the protocol by Szurdoki et al. (1996) with some modifications. Glutaraldehyde (GA) solution 50 % (W/V) (Sigma-Aldrich, Cat Nº 340855) was used as the cross-linker reagent while BSA (Sigma-Aldrich, Cat Nº A9085) was used as the carrier protein. Specifically, BSA (5 mg/mL) was dialyzed in 20 mM sodium phosphate buffer pH 6.0. A total of 10 µl of GA 50% (W/V) were then incubated with 1 mL of the dialyzed BSA solution overnight at room temperature. After incubation, excess GA was removed by dialyzing in Phosphate-buffered saline (PBS), followed by addition of 1 mg of FB1 (Santa Cruz Biotechnology, Cat Nº sc-201395A) to achieve the molar ratio of 20:1 (FB1:BSA-GA). The mixture was incubated at 4°C overnight on a Dynal Biotech rotary shaker (10 rpm) before the addition of 80 µl of glycine 1 M (Bio Basic, Cat Nº GB0235) to block unreacted aldehyde groups. The reaction mixture was further incubated at room temperature for 4 hours. Subsequently, sodium borohydride powder (Sigma-Aldrich, Cat Nº 452882) was added to the mixture (final concentration of 10 mg/mL) and incubated for 4 hours at room temperature. The obtained solution was then dialyzed and concentrated in 10 mM Borat buffer pH 8.5 using a 10 kDa Amicon® Ultra-4 Centrifugal Filter Unit (Millipore, Cat Nº UFC801024). Lastly, FB1-BSA conjugate was quantified using Nano Drop 2000 (Thermo Fisher Scientific) and stored at 4 °C.\n\nAnimal procedures. All animal procedures were approved by the Research Ethics Committee of Vietnam Military Medical University. All efforts were made to ameliorate harm to the animals, by conforming to the Principles of animal care and use in research adopted by the Vietnam Military Medical University.\n\nA total of two Fayoumi hens (aged 20 weeks) were sourced from Thuy Phuong Poultry Research Center, Vietnam for IgY production. Hens were housed individually in standard battery cages (800 cm2/hen) and received commercial rations (A55, Anova Feed) and water ad libitum. The temperature was kept between 25 and 35°C and the light cycle was 16 hours light/8 hours dark.\n\nPolyclonal IgY antibody against FB1-KLH was obtained as described previously (Do et al., 2016). Briefly, FB1-KLH was prepared according to procedures described by Szurdoki et al. (1996). Glutaraldehyde (GA) solution 50 % (W/V) (Sigma-Aldrich, Cat Nº 340855) was used as the cross-linker reagent. A total of 10 mg of KLH (Thermo Fisher Scientific, Cat Nº 77600) was dissolved in 12 mL of water and dialyzed against 2 L of 0.2% GA in 0.01 M PBS (pH 7.5) for 20 hours. Excess GA was removed by dialyzing in PBS, followed by dropwise addition of 2 mg of FB1 (Santa Cruz Biotechnology, Cat Nº sc-201395A). The mixture was incubated at 4°C overnight on a Dynal Biotech rotary shaker (10 rpm) before the addition of 80 µl of glycine 1 M (Bio Basic, Cat Nº GB0235) to block unreacted aldehyde groups. The reaction mixture was further incubated at room temperature for 4 hours. The obtained solution was then dialyzed and concentrated in PBS pH 7.5 using a 100 kDa Amicon® Ultra-4 Centrifugal Filter Unit (Millipore, Cat Nº UFC810024). Lastly, FB1-KLH conjugate was quantified using Nano Drop 2000 (Thermo Fisher Scientific) and stored at 4 °C.\n\nThe chickens were intramuscularly immunized three times in 10 days intervals to elicit strong immune response. For the first immunization, an injection dose of 1.0 mL was prepared by mixing 0.2 mg of FB1-KLH with an equal volume of complete Freund's adjuvant (Sigma-Aldrich, Cat Nº F5881). For the two subsequent booster immunizations, the amount of immunogen was decreased to 0.1 mg of FB1-KLH and incomplete Freund's adjuvant (Sigma-Aldrich, Cat Nº F5506) was used. Eggs were collected two weeks after the last immunization and stored at 4°C. The extraction of IgY was performed by polyethylene glycol (PEG) (Sigma-Aldrich, Cat Nº 81255) precipitation as described by Pauly et al. (2011). The eggshell was carefully cracked, and the yolk was transferred to a “yolk spoon” and filter paper to remove egg white. The egg yolk skin membrane was cut before the yolk was poured into a 50 ml tube. Twice the egg yolk volume of PBS was added to the tube and mixed by vortexing. PEG 6000 was added to achieve the final concentration of 3.5 % (w/v) and the tube was vortexed and rolled for 10 minutes on a Dynal Biotech rotary shaker (30 rpm) before being centrifuged at 8000 × g, 4 °C for 10 minutes. The supernatant was subjected to filtration and then to precipitation of IgY by adding PEG 6000 (final concentration 12 % (w/v)). The tube was vortexed and centrifuged at 8000 × g, 4 °C for 30 minutes and the supernatant was discarded. The pellet was dissolved in 10 mL PBS and PEG 6000 was added to achieve the final concentration of 12 % (w/v). Subsequently, the tube was centrifuged at 8000 × g, 4 °C for 30 minutes. The pellet was dissolved in 5 mL of PBS and IgY was further purified by microfiltration via a 0.45 μm membrane and ultrafiltration using 100 kDa Amicon® Ultra-4 Centrifugal Filter Units (Millipore, Cat Nº UFC810008). Finally, IgY was stored at -80°C in small aliquots.\n\nIgY-gold nanoparticle conjugate was prepared using BioReady 40 nm Carboxyl Gold (NanoComposix, Cat Nº AUXR40-5M). Particularly, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (Sigma-Aldrich, Cat Nº 03449) and N-hydroxysulfosuccinimide (Sulfo-NHS) (Sigma Aldrich, Cat Nº 56485) at 10 mg/mL in H2O were freshly prepared before conjugations. Anti-FB IgY was dialyzed in Antibody purification buffer (10 mM potassium phosphate, pH 7.4) using Amicon Ultra-0.5 Centrifugal Filter Unit (Millipore, Cat Nº UFC501096). One milliliter (0.83 mg) of BioReady 40 nm Carboxyl Gold was mixed with 20 µl and 40 µl of the prepared EDC and Sulfo-NHS respectively. The mixture was then incubated on a Dynal Biotech rotary shaker (15 rpm) at room temperature for 30 minutes then centrifuged at 3600 × g for 10 minutes. The supernatant was then removed completely, and the gold nanoparticles were resuspended in 1 mL of Reaction Buffer (5 mM potassium phosphate, 0.5 % 20K MW PEG, pH 7.4). The mixture was then incubated with 50 µg of anti-FB IgY on a Dynal Biotech rotary shaker (15 rpm) at room temperature for 2 hours. Subsequently, blocking of remaining NHS-esters was performed using 10 µl of 50% (w/v) hydroxylamine. IgY-conjugated gold nanoparticles were then washed three times with 1 mL of Reaction Buffer. Lastly, gold nanoparticle conjugate was resuspended in 10 mL of Conjugate Diluent (0.1X PBS, 0.5% BSA, 0.05% Sodium Azide) and stored at 4°C.\n\nTest strips were prepared following Posthuma-Trumpie et al. (2008) with some modifications. Briefly, a Linomat V (Camag, Cat Nº 022.7808) was used to dispense FB1-BSA and Mouse monoclonal 0.8C Anti-Chicken IgY H&L (Abcam, Cat Nº ab82229) at the test line and control line positions of a nitrocellulose membrane respectively. For the control line, immunoglobulins were dispensed at a dose of 0.5 µg/cm, at the position of 2 cm away from the dipping point. For the test line, FB1-BSA was dispensed at a dose of 1 µg/cm at the position of 1.5 cm away from the dipping point. The membrane was then dried for 2 hours at 37°C. A second plastic backing and an absorption pad (Extra Thick Blot Paper, BIO-RAD, Cat Nº 1703969) were applied; and the membranes were cut into 4 mm-wide test strips using an Autokun cutter (Hangzhou Autokun Technology). Test trips were sealed in aluminum packages with a desiccation pad and stored at 4°C until use. Three different membranes were tested, namely CNPC-SS12, 10 µm (MDI technologies, Cat Nº CNPC-SS12-10µm-25mm), UniSart® CN140 (Sartorius, Cat Nº 1UN14ER100025NTB), and UniSart® CN 95 (Sartorius, Cat Nº 1UN95ER100040WS).\n\nBlank and naturally contaminated maize grains were collected from local markets in Hanoi, Vietnam during the year of 2017. The samples were finely ground using an A 11 basic Analytical mill (IKA) and a 500 µm sieve.\n\nSpiking of FBs into maize was performed on a blank sample. Briefly, 5 g of ground maize were spiked with 10–40 µl of FB1 or FB2 stock solution of 1 mg/mL to achieve final content of 2000 – 8000 µg/kg. Spiked samples were left 24 hours at 4°C. Extraction of FBs and LFIA analysis were performed as described below.\n\nThe protocol for FB extraction from naturally contaminated or spiked samples (Figure 1) was based on the work of Pietri & Bertuzzi (2011) and Lattanzio et al. (2012). Instead of using organic solvents, FB was extracted with 0.4 M phosphate buffer (PB) at pH 7.5 (Pietri & Bertuzzi, 2011). Specifically, 5 g of maize flour were mixed with 45 mL of PB and blended using a T10 basic ULTRA-TURRAX® (IKA) at the highest speed for 3 minutes. The blended samples were then allowed to settle for 3 minutes to recover the supernatant, which was further diluted 1:3, 1:5, 1:10 or 1:20 in Running Buffer (100 mM Borat Buffer, 0.5 % BSA, 0.05% Tween®-20, 0.02 % NaN3, pH 8.5). For LFIA analysis, 100 µl of the diluted extracts were dispensed into a 2-mL lyophilization glass vials and incubated with 174 ng, 436 ng or 697 ng (corresponding to 2, 5, 8 µl) of detection conjugate for 0 to 60 minutes before being flowed vertically onto LFIA test strips. After 25 minutes, results could be read with the naked eye or captured by a Perfection V600 scanner (Epson). Optical densities of test lines and control lines were digitalized to obtain signal values using ImageJ software (ver.1.47) (Schneider et al., 2012). GraphPad Prism 6.0 (GraphPad Software Inc.) was used to statistically analyze and graph the data. Unpaired, two-tailed t-tests were performed to determine statistical significance.\n\nA total of 5 g of maize were homogenized in 45 mL of phosphate buffer for 3 minutes. The mixture was allowed to settle for 3 minutes before collection of the supernatant, which was further diluted in running buffer. A hundred microliters of the diluted extract were used for lateral flow immunoassays (LFIA) analysis. After being incubated with detection conjugate at room temperature, samples were flowed onto LFIA strips. Results were read with the naked eye after the strips absorbed fluid completely.\n\nTo test the specificity of the developed assay, four following mycotoxins at 100-fold and 1000-fold of MRL (deoxynivalenol at 1750 ng/mL and 17500 ng/mL; ochratoxin A at 5 ng/mL and 50 ng/mL; aflatoxin B1 at 10 ng/mL and 100 ng/mL; and zearalenone at 350 ng/mL and 3500 ng/mL) were spiked into diluted extracts of blank samples. All of these mycotoxins were purchased from FERMENTEK Ltd (Cat Nº: 51481-10-8, 303-47-9, 1162-65-8, 17924-92-4). The subsequent LFIA analyses were performed as mentioned above.\n\nFBs in maize were quantified by the EN 13585:2001 standard method (CEN, 2001) with some modifications. Briefly, 5g of maize were mixed with 5 mL of methanol-water and blended for 5 minutes using a T10 basic ULTRA-TURRAX® (IKA). Maize extracts were then collected by centrifugation (500 × g, 10 minutes) and filtering (Whatman) and 1 mL aliquot of filtrates was loaded into a preconditioned Bond-Elut strong-anion-exchanging cartridge (Agilent, Cat Nº 14102017). After washing with methanol, elution was performed using 10 mL of methanol-acetic acid (99:1 v/v). The eluate was then evaporated under a stream of nitrogen, washed with 1 mL of methanol and evaporated again. Dried samples were reconstituted in 1 mL of methanol before HPLC-MS/MS analysis. HPLC injection (10 µl) was performed on a system consisting of a Shimadzu LC-20ADVP pump; a Symmetry HPLC column (150 mm × 3.0 mm i.d. × 3.5 µm) maintained at 30°C (Waters, 186000695); and a SCIEX Triple Quad™ 5500 mass spectrometer. The analytical separation was performed with water-acid formic (99.9-0.1, v/v) and acetonitrile as mobile phases A and B respectively. The gradient elution program began with an isocratic step of 80:20 A:B for 2 minutes and then increased linearly to 10:90 A:B over 5 minutes, which was maintained for 3 minutes, and returned to the starting condition. The condition was then held constant for 3 minutes. The flow rate was kept at 0.5 mL/min. The HPLC column effluent was pumped to the MS/MS system, with the electrospray ionization (ESI) probe operating in positive mode. The following parameters were used: capillary voltage, 5000 V; desolvation gas temperature, 450°C; ion source gas 1 and gas 2 pressure, 40 and 30 psi, respectively. Detection was carried out in multiple reaction monitoring (MRM) mode with two transitions for each compound. Nitrogen was used as the collision gas, and the collision cell pressure was 7 psi. The reference standards of FBs were purchased from LGC Standards (Cat Nº B-MYC0400-C and B-MYC0420-1).\n\n\nResults\n\nThe LFIA developed in the present study is based on the competitive format in which polyclonal IgY antibody, showing recognition specificity toward both FB1 and FB2 (Do et al., 2016), is conjugated to gold nanoparticle (see underlying data (Tran et al., 2019)). The labeled antibody was mixed with the sample extract in a glass vial, and the mixture was incubated to allow antigen-antibody complexes to form before flowing onto the nitrocellulose membrane which contains a test line and a control line. In our assay, FB1-BSA conjugate was immobilized on the test line while a secondary antibody against chicken IgY was coated on the control line. In a negative sample, the free detection antibody binds to the FB1-BSA conjugate immobilized on the test line, forming a visible line. An excess of the labeled antibody migrates to the control line and binds to the secondary antibody. As a result, a negative sample will form two visible lines on the nitrocellulose membrane. In a positive sample, FBs in the sample extract will react with all of the available binding sites of the antibody, thus preventing attachment of the detection antibody to the FB1-BSA conjugate on the test line. All of the detection conjugate will migrate to the control line and will form a visible line. Consequently, a positive sample will form only one line at the control zone.\n\nOptimization has been performed with FB1, the most common mycotoxin in maize, so that the samples with FB1 concentration equal to or beyond the maximum residue limit of 4000 µg/kg, will result in no visible line at the test zone. To this end, the effects of nitrocellulose membrane type, dilution factor of maize homogenates in running buffer, amount of detection conjugate, and the incubation time between sample extract and detection conjugate, on the test performance were evaluated.\n\nSelection of nitrocellulose membrane. Flow rate and protein-binding capacity of nitrocellulose membranes directly affect sensitivity and run time of a LFIA (O’Farrell, 2008). Generally, nitrocellulose membranes with a low flow rate will facilitate the formation of immunocomplexes at the test and control lines. However, it could lead to extended run times and false positive results (O’Farrell, 2008). In the present study, selection of nitrocellulose membrane was carried out by analyzing running buffer mixed with detection conjugate (negative controls) on three different nitrocellulose membranes. Figure 2 indicated that UniSart® CN140 (Sartorius) and CNPC-SS12, 10 μm (MDI technologies) produced higher signal intensities than UniSart® CN95 (Sartorius). Although the difference in signal intensity between UniSart® CN140 and CNPC-SS12, 10 μm was not statistically significant (p = 0.9209), sample uptake time was significantly lower on UniSart® CN140 (Figure 2B). Therefore, UniSart® CN140 from Sartorius was chosen for subsequent experiments.\n\n(A) Images of negative controls (0 µg/mL fumonisin group B (FB)) on three different nitrocellulose membranes; 1, UniSart® CN95 (Sartorius), 2, UniSart® CN140 (Sartorius); 3, CNPC-SS12 10 µm (MDI technologies); CL, control line; TL, test line. (B) Quantification of signal intensities and sample uptake time for each type of membrane. Sample uptake time is defined as the total time required for membranes to absorb fluid completely. T, test line signal; C, control line signal.\n\nOptimization of dilution factor of maize extract. Food sample extracts are commonly diluted before analysis by LFIA to minimize the negative effects of sample matrix on antibody-antigen reactions (Anfossi et al., 2011; Lattanzio et al., 2012). To determine the optimal dilution factor, blank maize grains were subjected to extraction using phosphate buffer (PB) and dilution in running buffer with ratios ranging from 1:3 to 1:20. According to Pietri & Bertuzzi (2011), average recovery percentages were 95.5±1.9% and 96.7±2.1% for FB1 and FB2 when extracted in PB. Furthermore, this extraction method does not require the use of toxic solvents and it may prevent possible inhibiting effects of organic solvents on antibody-antigen reaction (Rehan & Younus, 2006; Russell et al., 1989). Test line intensities generated by the diluted extract samples were compared with those from negative controls. Figure 3 revealed that signal intensities were decreased at dilution factors of 1:3 and 1:5, comparing to those from negative controls. However, starting from 1:10 dilution, test line signals were similar to negative controls (Figure 3B). As a result, an optimal dilution factor of 1:10 was set for subsequent experiments.\n\n(A) Images of diluted extracts of blank maize samples (negative with fumonisin group B (FB) by high performance liquid chromatography (HPLC)) with different dilution factors on representative lateral flow immunoassay strips; 1, 2, 3, 4, maize homogenates diluted 3-, 5-, 10-, 20-fold respectively in running buffer; 5, negative controls. CL, control line; TL, test line. (B) Quantification of test line signals. Control, negative control; AU, arbitrary unit.\n\nOptimization of amount of detection conjugate. Quantity of labeled antibody directly affects the limit of detection of a competitive LFIA. In fact, if a low amount of detection conjugate is used, no visible test lines will be formed even low levels of FBs (less than 4000 µg/kg) are present in the samples. Furthermore, using a low amount of detection conjugate will decrease the signal intensities at both test and control lines, causing difficulties in result interpretation. On the other hand, using an excessive amount of detection conjugate will negatively affect the analytical sensitivity of the assay as more toxins are required to saturate all the binding sites of the detection antibody.\n\nIn the present study, various amounts (174 ng, 436 ng, and 697 ng corresponding to 2, 5, and 8 µl) of detection conjugate were used to react with FB1 extracted from blank samples spiked with this toxin at 2000 µg/kg, 4000 µg/kg or 8000 µg/kg. For samples spiked with 2000 µg/kg FB1, test lines were observed on all test strips regardless of the amounts of detection conjugate used (Figure 4). Conversely, no test line was observed when FB1 is present at 8000 µg/kg (Figure 4). At the cut-off level of 4000 µg/kg, test line signal was still present when 697 ng of detection conjugate were used while no test line was visible when 174 ng or 436 ng of detection conjugate were used. However, using 174 ng of detection conjugate resulted in low signals of test line on negative controls (Figure 4). Therefore, 436 ng of IgY-conjugated gold nanoparticles were used for further studies.\n\n(A) Images of negative controls and diluted extracts of blank maize samples spiked with FB1 at 2000 µg/kg, 4000 µg/kg, and 8000 µg/kg using 174 ng, 436 ng or 697 ng of detection conjugate on representative lateral flow immunoassay (LFIA) strips. Maize extracts were incubated with detection conjugate for one hour at room temperature before being analyzed on LFIA strips. Experiment was performed with 8 replicates. CL, control line; TL, test line. (B) Quantification of test line signals. AU, arbitrary unit.\n\nOptimization of incubation time between samples and detection conjugate. Effects of incubation step between detection conjugate and FB1 at the cut-off level (extracted from blank samples spiked with FB1 at 4000 µg/kg) on the test performance were assessed by varying the incubation time from 0 to 60 minutes. Results (Figure 5) indicated that test lines were still visible when the incubation time was 0 or 15 minutes, while no test lines were observed when the incubation time was 30 or 60 minutes. To shorten the analytical procedure, an incubation time of 30 minutes was chosen.\n\n(A) Images of diluted extract (from blank sample spiked with fumonisin B1 (FB1) at 4000 µg/kg) incubated with 436 ng of detection conjugate for 0, 15, 30 and 60 minutes on representative lateral flow immunoassay strips. 1, negative control; 2, 3, 4, 5, incubation time of 0, 15, 30, 60 minutes respectively; CL, control line; TL, test line. (B) Quantification of signal intensities. NC, negative control; AU, arbitrary unit.\n\nPreviously, we have shown that the polyclonal IgY antibody used in the present study, recognized FB1 and FB2 with different affinities (IC50 = 10 and 49 ng/ml for FB1 and FB2 respectively) (Do et al., 2016). To determine if the developed LFIA could detect FB2 in maize at the cut-off level of 4000 µg/kg, this toxin was spiked into a blank sample at 2000 µg/kg, 4000 µg/kg, and 8000 µg/kg. Results (Figure 6) showed that no visible line was formed at test zone for samples spiked with 4000 µg/kg and 8000 µg/kg of FB2. On the contrary, faint signals were still observed at the test line for samples spiked with 2000 µg/kg of FB2. Therefore, the limit of detection of our LFIA for FB2 was also 4000 µg/kg, meaning that the developed test could be used for screening of total FBs in maize. The extended incubation time between detection conjugate and the toxins (30 minutes) and the optimal amount of labeled antibody are likely able to compensate for the difference in affinity of IgY antibody for FB1 and FB2.\n\n(A) Images of negative controls (1) and diluted extracts of blank maize sample spiked with FB2 at 2000 µg/kg (2), 4000 µg/kg (3), and 8000 µg/kg (4) on representative lateral flow immunoassay strips. CL, control line; TL, test line. Experiment was performed with 8 replicates. (B) Quantification of test line signals. AU, arbitrary unit.\n\nCross-reactivity tests were performed using deoxynivalenol, ochratoxin A, aflatoxin B1 and zearalenone spiked into blank maize extracts at two different concentration levels (100-fold and 1000-fold of MRL). Figure 7 showed that there was no difference in signal intensities at test line position between negative control and samples spiked with low and high concentrations of the tested mycotoxins. Therefore, the developed LFIA did not cross-react with deoxynivalenol, ochratoxin A, aflatoxin B1 and zearalenone.\n\nDeoxynivalenol (1750 and 17500 ng/mL), ochratoxin A (5 and 50 ng/mL), aflatoxin B1 (10 and 100 ng/mL), zearalenone (350 and 3500 ng/mL) were spiked into diluted extracts of blank maize samples and incubated with detection conjugate for 30 minutes before being analyzed on lateral flow immunoassay (LFIA) test strips. Negative, negative controls; Positive, 40 ng/mL fumonisin B1 (FB1), CL, control line; TL, test line.\n\nA total of 19 maize samples were analyzed by HPLC-MS and the developed assay. By HPLC-MS, all samples were negative with FB2, while 11 samples were positive with FB1, with concentrations ranging from 27 µg/kg to 7850 µg/kg (Table 1). One sample (No 16: 7850 µg/kg of FB1) exceeded the maximum legislative limits (EU) of 4000 µg/kg (CEN, 2001). Notably, only this sample was positive by IgY LFIA. Although the sample size was relatively low, these findings indicated that results by the IgY LFIA were in good agreement with those from HPLC-MS method as no false positive or false negative results were found.\n\n\nConclusions\n\nIn conclusion, this is the first study using polyclonal IgY antibody in LFIA for simple detection of FBs in maize with the limit of detection meeting the EU regulatory requirements for MRL of 4000 µg/kg. The main advantage of this assay is its cost-effectiveness as one single egg from inoculated hens yielded approximately 8 mg of purified IgY, which is enough to produce 320000 tests. One limitation of the developed LFIA is that the analysis times are longer than those of commercial assays as it requires an incubation step of 30 minutes to allow FBs to saturate the binding sites of detection antibody. However, as mentioned above, this additional step was likely able to compensate for the difference in affinity of IgY antibody for FB1 and FB2, enabling the new assay to meet the cut-off level of 4000 µg/kg for both these toxins.\n\n\nData availability\n\nFigshare: Raw data for \"Development of an IgY-based lateral flow immunoassay for detection of fumonisin B in maize\". https://doi.org/10.6084/m9.figshare.8320775 (Tran et al., 2019)\n\nThis project contains the following underlying data:\n\nDataset 1_Raw scans of lateral flow test strips.rar (Folder includes raw images of lateral flow strips)\n\nDataset 2.pptx (PowerPoint file includes HPLC-MS chromatograms of naturally contaminated samples for quantification of FB1 and FB2.)\n\nDataset 3.xlsx (Excel file includes quantification of signal intensities of lateral flow test strips and quantification of FB1 and FB2 from HPLC-MS output.)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nResearch reported in this publication was supported by the Hanoi Office of Science and Technology [01C-06/02-2015-2].\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank Trang Huyen Thi Tran and Trang Mai Ta for their support during the production of IgY.\n\n\nReferences\n\nAnfossi L, Calderara M, Baggiani C, et al.: Development and application of a quantitative lateral flow immunoassay for fumonisins in maize. Anal Chim Acta. 2010; 682(1–2): 104–109. PubMed Abstract \| Publisher Full Text\n\nAnfossi L, D'Arco G, Calderara M, et al.: Development of a quantitative lateral flow immunoassay for the detection of aflatoxins in maize. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2011; 28(2): 226–234. PubMed Abstract \| Publisher Full Text\n\nChilaka CA, De Boevre M, Atanda OO, et al.: Occurrence of Fusarium Mycotoxins in Cereal Crops and Processed Products (Ogi) from Nigeria. Toxins (Basel). 2016; 8(11): pii: E342. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDo NB, Tran THT, Ta MT, et al.: Obtaining high affinity IgY against fumonisin with potential use in immunodiagnostic. Vietnamese Journal of Military Pharmaco-medicine. 2016; 41(5): 29–34.\n\nEuropean Commission: Commission regulation (EC) No. 856/2005 of 6 June 2005 amending Regulation (EC) No. 466/2001 as regards Fusarium toxins. Official Journal of the European Union. 2005; L143: 3–8. Reference Source\n\nEuropean Committee for Standardization (CEN): CEN EN 13585 – Foodstuffs – Determination of fumonisins B1 and B2 in maize – HPLC method with solid phase extraction clean-up. European Committee for Standardization, Brussels, Belgium, 2001. Reference Source\n\nFu M, Li R, Guo C, et al.: Natural incidence of Fusarium species and fumonisins B1 and B2 associated with maize kernels from nine provinces in China in 2012. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2015; 32(4): 503–511. PubMed Abstract \| Publisher Full Text\n\nGandhi S, Caplash N, Sharma P, et al.: Strip-based immunochromatographic assay using specific egg yolk antibodies for rapid detection of morphine in urine samples. Biosens Bioelectron. 2009; 25(2): 502–505. PubMed Abstract \| Publisher Full Text\n\nGelderblom WC, Jaskiewicz K, Marasas WF, et al.: Fumonisins--novel mycotoxins with cancer-promoting activity produced by Fusarium moniliforme. Appl Environ Microbiol. 1988; 54(7): 1806–1811. PubMed Abstract \| Free Full Text\n\nGetachew A, Chala A, Hofgaard IS, et al.: Multimycotoxin and fungal analysis of maize grains from south and southwestern Ethiopia. Food Addit Contam Part B Surveill. 2018; 11(1): 64–74. PubMed Abstract \| Publisher Full Text\n\nGuo C, Liu Y, Jiang Y, et al.: Fusarium species identification and fumonisin production in maize kernels from Shandong Province, China, from 2012 to 2014. Food Addit Contam Part B Surveill. 2016; 9(3): 203–209. PubMed Abstract \| Publisher Full Text\n\nHieu Phuong N, Quang Thieu N, Ogle B, et al.: Aflatoxins, Fumonisins and Zearalenone Contamination of Maize in the Southeastern and Central Highlands Provinces of Vietnam. 2015; 5(4): 1195–1203. Publisher Full Text\n\nHu L, Liu H, Yang J, et al.: Free and hidden fumonisins in raw maize and maize-based products from China. Food Addit Contam Part B Surveill. 2019; 12(2): 90–96. PubMed Abstract \| Publisher Full Text\n\nJackson LS, Hlywka JJ, Senthil KR, et al.: Effect of thermal processing on the stability of fumonisins. Adv Exp Med Biol. 1996; 392: 345–353. PubMed Abstract \| Publisher Full Text\n\nJin W, Yamada K, Ikami M, et al.: Application of IgY to sandwich enzyme-linked immunosorbent assays, lateral flow devices, and immunopillar chips for detecting staphylococcal enterotoxins in milk and dairy products. J Microbiol Methods. 2013; 92(3): 323–331. PubMed Abstract \| Publisher Full Text\n\nKamala A, Kimanya M, Haesaert G, et al.: Local post-harvest practices associated with aflatoxin and fumonisin contamination of maize in three agro ecological zones of Tanzania. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2016; 33(3): 551–559. PubMed Abstract \| Publisher Full Text\n\nKovacs-Nolan J, Mine Y: Avian egg antibodies: basic and potential applications. Avian Poult Biol Rev. 2004; 15(1): 25–46. Reference Source\n\nLattanzio VM, Nivarlet N, Lippolis V, et al.: Multiplex dipstick immunoassay for semi-quantitative determination of Fusarium mycotoxins in cereals. Anal Chim Acta. 2012; 718: 99–108. PubMed Abstract \| Publisher Full Text\n\nLiu Y, Jiang Y, Li R, et al.: Natural occurrence of fumonisins B1 and B2 in maize from eight provinces of China in 2014. Food Addit Contam Part B Surveill. 2017; 10(2): 113–117. PubMed Abstract \| Publisher Full Text\n\nMngqawa P, Shephard GS, Green IR, et al.: Mycotoxin contamination of home-grown maize in rural northern South Africa (Limpopo and Mpumalanga Provinces). Food Addit Contam Part B Surveill. 2016; 9(1): 38–45. PubMed Abstract \| Publisher Full Text\n\nMunkvold GP, Arias S, Taschl I, et al.: Chapter 9 - Mycotoxins in Corn: Occurrence, Impacts, and Management. In S. O. Serna-Saldivar (Ed.), Corn (Third Edition). Oxford: AACC International Press. 2019; 235–287. Publisher Full Text\n\nMutiga SK, Hoffmann V, Harvey JW, et al.: Assessment of Aflatoxin and Fumonisin Contamination of Maize in Western Kenya. Phytopathology. 2015; 105(9): 1250–1261. PubMed Abstract \| Publisher Full Text\n\nMwalwayo DS, Thole B: Prevalence of aflatoxin and fumonisins (B1 + B2) in maize consumed in rural Malawi. Toxicol Rep. 2016; 3: 173–179. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNTP: NTP Technical Report on the Toxicology and Carcinogenesis Studies of Fumonisin B1 (CAS No. 116355-83-0) in F344/N Rats and B6C3F1 Mice (Feed Studies). Research Triangle Park, NC: National Toxicology Program, U.S. Department of Health and Human Services, National Institutes of Health (NTP Technical Report No. 496; NIH Publication No. 99-3955). 2001. Reference Source\n\nO’Farrell B: Evolution in Lateral Flow–Based Immunoassay Systems. Lateral Flow Immunoassay. 2008; 1–33. Publisher Full Text\n\nPauly D, Chacana PA, Calzado EG, et al.: IgY technology: extraction of chicken antibodies from egg yolk by polyethylene glycol (PEG) precipitation. J Vis Exp. 2011; (51): pii: 3084. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPietri A, Bertuzzi T: Simple Phosphate Buffer Extraction for the Determination of Fumonisins in Masa, Maize, and Derived Products. Food Anal Methods. 2011; 5(5): 1088–1096. Publisher Full Text\n\nPosthuma-Trumpie GA, Korf J, van Amerongen A: Development of a competitive lateral flow immunoassay for progesterone: influence of coating conjugates and buffer components. Anal Bioanal Chem. 2008; 392(6): 1215–1223. PubMed Abstract \| Publisher Full Text\n\nRehan M, Younus H: Effect of organic solvents on the conformation and interaction of catalase and anticatalase antibodies. Int J Biol Macromol. 2006; 38(3–5): 289–295. PubMed Abstract \| Publisher Full Text\n\nRheeder JP, Marasas WF, Vismer HF: Production of fumonisin analogs by Fusarium species. Appl Environ Microbiol. 2002; 68(5): 2101–2105. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRussell AJ, Trudel LJ, Skipper PL, et al.: Antibody-antigen binding in organic solvents. Biochem Biophys Res Commun. 1989; 158(1): 80–85. PubMed Abstract \| Publisher Full Text\n\nSchneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012; 9(7): 671–675. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScussel VM, Savi GD, Costas LL, et al.: Fumonisins in corn (Zea mays L.) from Southern Brazil. Food Addit Contam Part B Surveill. 2014; 7(2): 151–155. PubMed Abstract \| Publisher Full Text\n\nSzurdoki F, Trousdale E, Ward B, et al.: Synthesis of Protein Conjugates and Development of Immunoassays for AAL Toxins. J Agric Food Chem. 1996; 44(7): 1796–1803. Publisher Full Text\n\nTran VT, Do NB, Nguyen TPT, et al.: Raw data for \"Development of an IgY-based lateral flow immunoassay for detection of fumonisin B in maize\". figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8320775.v4\n\nVenkataramana M, Navya K, Chandranayaka S, et al.: Development and validation of an immunochromatographic assay for rapid detection of fumonisin B1 from cereal samples. J Food Sci Technol. 2014; 51(9): 1920–1928. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang YK, Shi YB, Zou Q, et al.: Development of a rapid and simultaneous immunochromatographic assay for the determination of zearalenone and fumonisin B1 in corn, wheat and feedstuff samples. Food Control. 2013; 31(1): 180–188. Publisher Full Text\n\nWang Z, Li H, Li C, et al.: Development and application of a quantitative fluorescence-based immunochromatographic assay for fumonisin b1 in maize. J Agric Food Chem. 2014; 62(27): 6294–6298. PubMed Abstract \| Publisher Full Text\n\nWielogorska E, Mooney M, Eskola M, et al.: Occurrence and Human-Health Impacts of Mycotoxins in Somalia. J Agric Food Chem. 2019; 67(7): 2052–2060. PubMed Abstract \| Publisher Full Text\n\nYamada K, Wanchun J, Ohkura T, et al.: Detection of methicillin-resistant Staphylococcus aureus using a specific anti-PBP2a chicken IgY antibody. Jpn J Infect Dis. 2013; 66(2): 103–108. PubMed Abstract \| Publisher Full Text\n\nYazar S, Omurtag GZ: Fumonisins, trichothecenes and zearalenone in cereals. Int J Mol Sci. 2008; 9(11): 2062–2090. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYu Q, Li H, Li C, et al.: Gold nanoparticles-based lateral flow immunoassay with silver staining for simultaneous detection of fumonisin B1 and deoxynivalenol. Food Control. 2015; 54: 347–352. Publisher Full Text\n\nZhang Y, Kong H, Liu X, et al.: Quantum dot-based lateral-flow immunoassay for rapid detection of rhein using specific egg yolk antibodies. Artif Cells Nanomed Biotechnol. 2018; 46(8): 1685–1693. PubMed Abstract \| Publisher Full Text" }	[ { "id": "51089", "date": "14 Aug 2019", "name": "Venkataramana Mudili", "expertise": [ "Reviewer Expertise Toxicology and Immunology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present manuscript submitted by Tien et al. is a very interesting study in current food contaminants aspect. However, my main criticism goes to the detection limits of assay and the target selection in the study. As Fumonisins are a group of toxins, out of which fumonisin was target in the present study was not clear, for example among the fumonisin FB1 is more potent and frequently reported one in may kind of food grains mostly in wheat and maize and the cross reactivity patterns with group of Fusarium toxins need to be studied for example DON, T-2 toxin, Nivalenol etc.\nIntroduction part is too vague, need to be improved with recent and relevant references.\n\nPreparation of IgY-conjugated gold nano particles is not clear, it's totally confusing, authors should clear draft the protocol in a supplementary section or main text.\n\nEthical committee guidelines and permission to carry out the study need be given the manuscript.\n\nSelection of nitrocellulose membrane: its very crucial in developing the LFA assay, in the present study sufficient information was not presented by the authors in this section. please be give the clear protocol how its been chosen.\n\nOD 4000ug/kg is very high, many studies reported that the LD is under nano gram levels, this need to be further improved for their sensitivity.\n\nOver study looks good and can be indexed in this journal after substantial revision of the manuscript.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5065", "date": "12 Dec 2019", "name": "Tung Tran", "role": "Author Response", "response": "General comments:We thank the reviewer for comments and detailed suggestions. We herein clarify issues shown by the reviewer. The manuscript has been rewritten according to the comments, and a discussion part has been added to further explain the need for a new lateral flow immunoassay (LFIA) for total fumonisins (defined as the sum of FB1 and FB2) with the limit of detection (LOD) equal to the maximum residue limit (MRL) of this mycotoxin group in raw maize and to underline the advantages of IgY for the production of LFIA. As Fumonisins are a group of toxins, which fumonisin was target in the present study was not clear, for example, among the fumonisin, FB1 is more potent and frequently reported one in many kinds of food grains, mostly in wheat and maize. - In the present study, the targets are Fumonisin B1 and Fumonisin B2 (FB1 and FB2) as they are the most frequently reported fumonisins in foods in general, and in raw maize in particular (Abbas, 2006; D’Arco et al., 2009; Magro et al., 2011; Yazar & Omurtag, 2008)- Besides, according to the Regulation No 1126/2007 of the Commission of the European communities, the maximum residue limit of fumonisins in raw maize (defined as sum of FB1 and FB2) is fixed at 4000 µg/kg (EC, 2007). The cross reactivity patterns with group of Fusarium toxins need to be studied for example DON, T-2 toxin, Nivalenol etc. - In the manuscript, we have showed an absence of cross-reactivity of the developed test with deoxynivalenol, ochratoxin A, aflatoxin B1 and zearalenone.- DON, T-2 toxin, Nivalenol, which were suggested by the reviewer, belong to the same group of trichothecenes, and have similar structure (McCormick et al., 2011). Therefore, we performed only the cross-reactivity test using DON, which has been shown to be the most prevalent trichothecenes in cereals (Foroud et al., 2019; Payros et al., 2016). Introduction part is too vague, need to be improved with recent and relevant references. - We thank the reviewer for raising the point, the introduction has been modified and updated with recent literatures. Preparation of IgY-conjugated gold nano particles is not clear, it's totally confusing, authors should clear draft the protocol in a supplementary section or main text. - The principle of conjugate preparation has been added to the manuscript and the protocol has been rewritten for better clarity. Ethical committee guidelines and permission to carry out the study need to be given the manuscript. - We have provided, in this revision of the manuscript, an ethics approval for animal use for this study. Selection of nitrocellulose membrane: it’s very crucial in developing the LFA assay, in the present study, sufficient information was not presented by the authors in this section. please be give the clear protocol how it’s been chosen. - We thank the reviewer for raising the point. Details on flow rates of the membranes have been added as follows:“CNPC-SS12, 10 µm with wicking time of 140 ± 28s/40 mm (MDI technologies, Cat Nº CNPC-SS12-10µm-25mm), UniSart® CN140 with wicking time of 95-155s/ 40 mm (Sartorius, Cat Nº 1UN14ER100025NTB), and UniSart® CN 95 with wicking time of 65-115s/ 40 mm (Sartorius, Cat Nº 1UN95ER100040WS)”.- Furthermore, according to the manufacturers, CNPC-SS12-10µm is recommended for environmental and agriculture analytes; CN95 is recommended when a quick response is desirable; whereas CN140 is recommended when high sensitivity is required. LOD 4000ug/kg is very high, many studies reported that the LOD is under nano gram levels, this need to be further improved for their sensitivity. - We thank the reviewer for raising this point. It is certainly feasible to optimize the assay to achieve a lower limit of detection. However, the LOD was set at 4000 µg/kg as it is the maximum residue limit (MRL) adopted by the Commission of the European communities. Similar to other lateral flow immunoassays for detection of mycotoxins in foods, this assay is intended for quick screening, and positive results need to be confirmed by instrumental analytical techniques, such as HPLC, LC/MS. Consequently, if the sensitivity of the assay is in the range of nanograms, we will obtain a lot of false-positive results (according to the adopted MRL) that will be revealed by costly confirmatory methods. This will increase the cost of the whole analytical procedure. ReferencesAbbas, H. K. (2006). Aflatoxin and fumonisin contamination of corn (maize, Zea mays) hybrids in Arkansas. Crop protection, v. 25(no. 1), pp. 1-9-2006 v.2025 no.2001.D’Arco, G., Fernández-Franzón, M., Font, G., Damiani, P., & Mañes, J. (2009). Survey of fumonisins B1, B2 and B3 in conventional and organic retail corn products in Spain and Italy and estimated dietary exposure. Food Additives & Contaminants: Part B, 2(2), 146-153. doi:10.1080/02652030903148314EC, European Commission (2007). Commission Regulation (EC) no 1126/2007 of 28 September 2007 setting maximum levels for certain contaminants in foodstuffs as regards Fusarium toxins in maize and maize products. Official Journal of the European Union, L, 255,14–17.Foroud, N. A., Baines, D., Gagkaeva, T. Y., Thakor, N., Badea, A., Steiner, B., et al. (2019). Trichothecenes in Cereal Grains – An Update. 11(11), 634.Magro, S. L., Campaniello, M., Nardiello, D., & Muscarella, M. (2011). Assessment of Fumonisins B1 and B2 Levels in Commercial Maize-Based Food Products by Liquid Chromatography with Fluorimetric Detection and Postcolumn Chemical Derivatization. J Food Sci, 76(1), T1-T4. doi:10.1111/j.1750-3841.2010.01948.xMcCormick, S. P., Stanley, A. M., Stover, N. A., & Alexander, N. J. (2011). Trichothecenes: from simple to complex mycotoxins. Toxins, 3(7), 802-814. doi:10.3390/toxins3070802Payros, D., Alassane-Kpembi, I., Pierron, A., Loiseau, N., Pinton, P., & Oswald, I. P. (2016). Toxicology of deoxynivalenol and its acetylated and modified forms. Arch Toxicol, 90(12), 2931-2957. doi:10.1007/s00204-016-1826-4Yazar, S., & Omurtag, G. Z. (2008). Fumonisins, trichothecenes and zearalenone in cereals. International journal of molecular sciences, 9(11), 2062-2090. doi:10.3390/ijms9112062" } ] }, { "id": "52437", "date": "23 Aug 2019", "name": "Zhanhui Wang", "expertise": [ "Reviewer Expertise Analytical Chemistry", "Food Safety", "Antibody production and immunoassay." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper described a an IgY-based lateral flow immunoassay for detection of fumonisin B in maize. In general, the paper is more like an experimental report than scientific study in my opinion. The paper provided little new information for readers and the commercial products of LFIA for FBs are already available in the market with high performances. All techniques used in the study are conventional and no any improvement was achieved.\n\nThe LFIAs for the rapid detection of FBs are reported by using many probes not only gold. The performance of the LFIA developed by the authors in term of sensitivity, specificity and accuracy are not comparable with those of reports.\n\nThe novelty of the study relied on the first report of usage of IgY in LFIA for FB in my opinion, however, the production of IgY to FB already reported by the authors. The paper should show the advantages and disadvantages of usage of IgY in LFIA. And there are no comparative data with other antibodies like antibody from mouse or rabbit. Exactly, the production of IgY to FB is also a well established techniques.\n\nThe main body of the manuscript is the optimization of LFIA conditions, the procedure of optimization is necessary for any analytical methods and not the point of the study.\n\nI do not think the IgY used in the study is the first choice since the affinity and specificity of IgY is inferior in comparison with reported antibodies, which may result in inaccuracy determination.\n\nThe authors should provide the confirmation data for protein conjugates, gold conjugates if they are firstly reported or just cited the reference if these data already reported. For LFIAs, the gold-antibody is important, why the authors have not optimized the preparation of gold-protein conjugates? The low sensitivity of the LFIAs maybe derived from the coating antigens and suggest the authors should evaluate different coating antigens differentiating with ratio, hapten and conjugation methods. In addition, the authors should test the cross-reactivity with FB3 since the analog is available and a potential interferent.\n\nIn conclusion, the paper did not describe a new experimental, observational, or computational method, test or procedure. I have to be against indexing and hope the suggestions could help the authors improve the study.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "5066", "date": "12 Dec 2019", "name": "Tung Tran", "role": "Author Response", "response": "General comments:We thank the reviewer for comments and detailed suggestions. Please find below our answers for the points raised by the reviewer. The manuscript has been rewritten according to the comments, and a discussion part has been added to further explain the need for a new lateral flow immunoassay (LFIA) for total fumonisins (defined as the sum of FB1 and FB2) with the limit of detection (LOD) equal to the maximum residue limit (MRL) of this mycotoxin group in raw maize and to underline the advantages of IgY for the production of LFIA. The paper described an IgY-based lateral flow immunoassay for detection of fumonisin B in maize. In general, the paper is more like an experimental report than scientific study in my opinion. The paper provided little new information for readers and the commercial products of LFIA for FBs are already available in the market with high performances. All techniques used in the study are conventional and not any improvement was achieved. - We agree that commercial Lateral flow immunoassays (LFIAs) for fumonisins B are available. However, most of which are based on monoclonal antibodies that are costly to produce. Several commercial LFIAs for fumonisins require a reader to obtain accurate results, which limits their applicability in low-resourced environments.- In this study, we developed a new polyclonal IgY-based LFIA, which significantly reduces the cost of production. Furthermore, result interpretation is based on complete test line disappearance, instead of a decreased intensity of test line color. Thus, results could be read with the naked eye.- Another novelty of this study is that the developed LFIA was optimized to have the limit of detection for total fumonisins equal to the maximum residue limit adopted by the Commission of the European Communities, which would reduce the false positive rate comparing to other LFIAs reported in the literature, while maintaining the simplicity of the assay as the results could be read with the naked eye The LFIAs for the rapid detection of FBs are reported by using many probes not only gold. The performance of the LFIA developed by the authors in term of sensitivity, specificity, and accuracy are not comparable with those of reports. - We agree that other research groups have successfully applied probes other than gold nanoparticles, which increased the sensitivity of their LFIAs. However, in this case, the developed test is intended for screening of raw maize with LOD equal to the MRL (4000 µg/kg for the sum of FB1 and FB2) adopted by the Commission of the European communities. Of note, this maximum level is particularly high compared to MRLs of other mycotoxins in cereals (5 µg/kg for ochratoxin A, 4 µg/kg for total aflatoxins…) (EC, 2006). For that reason, we chose gold nanoparticles, which have high stability, as probes (Guerrini et al., 2018; Sajid et al., 2015).- In terms of accuracy, we have performed analyses of naturally contaminated maize samples by both the developed IgY LFIA and HPLC-MS/MS method and found perfect agreement between the two assays. One positive sample by LFIA was confirmed, by HPLC-MS/MS, to be contaminated with FB1 at the level higher than 4000 µg/kg. The novelty of the study relied on the first report of usage of IgY in LFIA for FB in my opinion, however, the production of IgY to FB already reported by the authors. The paper should show the advantages and disadvantages of usage of IgY in LFIA. And there are no comparative data with other antibodies like antibody from mouse or rabbit. Exactly, the production of IgY to FB is also a well established techniques. - Indeed, this is the first report of application of IgY in development of LFIA for total fumonisins. In our previous study, IC50 values of the obtained IgY were 10 and 49 ng/mL for FB1 and FB2 respectively (Do et al., 2016). These results show that the affinities of our IgY towards these mycotoxins are comparable to or even higher than some monoclonal antibodies reported in the literature (Ling et al., 2014; Maragos et al., 2018; Yu & Chu, 1999; Zhang et al., 2018).- As mentioned in the manuscript, the main advantage of using IgY in LFIAs for total fumonisins is cost-effectiveness. From one egg of the immunized hen, we obtained approximately 8 mg of purified IgY, which is enough to produce 320000 tests. The main body of the manuscript is the optimization of LFIA conditions, the procedure of optimization is necessary for any analytical methods and not the point of the study. - The aim of the present study is to develop a new LFIA for total fumonisins with LOD equal to MRL of these toxins in raw maize, whose performance, LOD and cross-reactivity pattern rely on selection and optimization of test components. Therefore, it is necessary to perform and report optimization experiments. - Other researchers also demonstrated optimization results in their reports of development of LFIAs for detection of fumonisins (Anfossi et al., 2010; Hao et al., 2018; Wang et al., 2013) I do not think the IgY used in the study is the first choice since the affinity and specificity of IgY is inferior in comparison with reported antibodies, which may result in inaccuracy determination. - Once again, with the MRL at 4000 µg/kg, we do not need a test with very low LOD.- In term of test’s accuracy, mycotoxins are low-molecular weight molecules, thus, they need to be conjugated with carrier proteins to act as immunogens. Therefore, cross-reactivity with other mycotoxins is not possible, even though certain levels of mycotoxins may be present in the chicken feeds.- Our unpublished data also showed an absence of cross-reactivity of IgY against BSA as KLH-FB1 was used for antibody production while BSA-FB1 was immobilized on LFIA membranes. The authors should provide the confirmation data for protein conjugates, gold conjugates if they are firstly reported or just cited the reference if these data already reported. For LFIAs, the gold-antibody is important, why the authors have not optimized the preparation of gold-protein conjugates? The low sensitivity of the LFIAs maybe derived from the coating antigens and suggest the authors should evaluate different coating antigens differentiating with ratio, hapten and conjugation methods. In addition, the authors should test the cross-reactivity with FB3 since the analog is available and a potential interferent. - We agree with the reviewer that optimization of gold nanoparticle-antibody is a crucial step for increasing the sensitivity of LFIA, which is relevant in developing an assay that detect contaminants with low MRL, such as Aflatoxin. However, in this study, we aimed to develop a LFIA that detect fumonisins with LOD of 4000 µg/kg, therefore, gold nanoparticle-antibody conjugation was performed as recommended by the manufacturer to ensure the stability of the conjugate.- We agree that cross-reactivity with FB3 should be tested. However, we currently do not have access to standards of FB3. Additionally, all LFIAs reported in the literature did not evaluate the cross-reactivity with FB3. Furthermore, according to the Commission of the European communities, the maximum residue limit of fumonisins in raw maize accounts for the sum of FB1 and FB2 only. Anfossi, L., Calderara, M., Baggiani, C., Giovannoli, C., Arletti, E., & Giraudi, G. (2010). Development and application of a quantitative lateral flow immunoassay for fumonisins in maize. Anal Chim Acta, 682(1-2), 104-109. doi:10.1016/j.aca.2010.09.045 Guerrini, L., Alvarez-Puebla, R. A., & Pazos-Perez, N. (2018). Surface Modifications of Nanoparticles for Stability in Biological Fluids. Materials (Basel, Switzerland), 11(7), 1154. doi:10.3390/ma11071154 Hao, K., Suryoprabowo, S., Hong, T., Song, S., Liu, L., Zheng, Q., et al. (2018). Immunochromatographic strip for ultrasensitive detection of fumonisin B1. Food and Agricultural Immunology, 29(1), 699-710. doi:10.1080/09540105.2018.1439455 Ling, S., Pang, J., Yu, J., Wang, R., Liu, L., Ma, Y., et al. (2014). Preparation and identification of monoclonal antibody against fumonisin B(1) and development of detection by Ic-ELISA. Toxicon, 80, 64-72. doi:10.1016/j.toxicon.2013.12.008 Maragos, C. M., Sieve, K. K., & Busman, M. (2018). Development of antibodies for N-(1-deoxy-D-fructos-1-yl) fumonisin B1 and cross-reaction with modified fumonisins. World Mycotoxin Journal, 11(4), 493-502. doi:10.3920/WMJ2018.2308 Sajid, M., Kawde, A.-N., & Daud, M. (2015). Designs, formats and applications of lateral flow assay: A literature review. Journal of Saudi Chemical Society, 19(6), 689-705. doi:https://doi.org/10.1016/j.jscs.2014.09.001 Wang, Y., Shi, Y.-B., Zou, Q., Sun, J.-H., Chen, Z.-F., Wang, H.-a., et al. (2013). Development of a rapid and simultaneous immunochromatographic assay for the determination of zearalenone and fumonisin B1 in corn, wheat and feedstuff samples. Food Control, 31, 180–188. doi:10.1016/j.foodcont.2012.09.048 Yu, F.-Y., & Chu, F. S. (1999). Production and Characterization of Monoclonal Antibodies against Fumonisin B1. Food and Agricultural Immunology, 11(4), 297-306. doi:10.1080/09540109999681 Zhang, X., Wang, Z., Fang, Y., Sun, R., Cao, T., Paudyal, N., et al. (2018). Antibody Microarray Immunoassay for Simultaneous Quantification of Multiple Mycotoxins in Corn Samples. Toxins, 10(10), 415. doi:10.3390/toxins10100415" } ] }, { "id": "52435", "date": "02 Sep 2019", "name": "Boris B. Dzantiev", "expertise": [ "Reviewer Expertise immunoassays", "immunosensors", "immunochromatography", "nanoparticles" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe first IgY-based lateral flow immunoassay of fumonisin B is presented. The earlier known techniques were based on the IgG use. At whole, the study is well-described and contains all necessary stages of the assay optimization, characterization and validation. However, the necessity of new test is not clear from the manuscript. The common advantages of IgY are widely known, but were not demonstrated for the proposed test system. To clarify this question, the data about reached advantages (lower reactants consumption, higher stability, etc.) of the proposed test in comparison with the known IgG-based test strips for fumonisin B should be added with their experimental / quantitative confirmation.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "5067", "date": "12 Dec 2019", "name": "Tung Tran", "role": "Author Response", "response": "We thank the reviewer for comments and detailed suggestions. We herein clarify issues shown by the reviewer. The manuscript has been rewritten according to the comments, and a discussion part has been added to further explain the need for a new lateral flow immunoassay (LFIA) for total fumonisins (defined as the sum of FB1 and FB2) with the limit of detection (LOD) equal to the maximum residue limit (MRL) of this mycotoxin group in raw maize and to underline the advantages of IgY for the production of LFIA. The first IgY-based lateral flow immunoassay of fumonisin B is presented. The earlier known techniques were based on the IgG use. At whole, the study is well-described and contains all necessary stages of the assay optimization, characterization and validation. However, the necessity of new test is not clear from the manuscript. The common advantages of IgY are widely known, but were not demonstrated for the proposed test system. To clarify this question, the data about reached advantages (lower reactants consumption, higher stability, etc.) of the proposed test in comparison with the known IgG-based test strips for fumonisin B should be added with their experimental / quantitative confirmation. - The affinities of polyclonal IgY used in this study (IC50 = 10 and 49 ng/mL for FB1 and FB2) towards FB1 and FB2 are comparable to monoclonal antibodies reported in the literature (Ling et al., 2014; Maragos et al., 2018; Yu & Chu, 1999; Zhang et al., 2018). Therefore, there is likely no difference in reactant consumption between producing IgY-based and IgG-based LFIAs for fumonisin detection. However, IgY can be produced in large quantity at a much lower cost. We obtained approximately 8 mg of purified IgY from one egg, which is enough to produce 320000 tests.- IgG has been shown to be more stable than IgY under acid, heat-treatment, and Guanidine-HCl denaturation (Shimizu et al., 1992). However, from our experiences, the IgY polyclonal antibody in this study was produced in 2016, stored at -80 oC, and used for the development of the assay during the period of 2018-2019. This shows that polyclonal IgY antibody was quite stable over extended periods of time under preservation at -80oC.- On the stability of the IgY-gold nanoparticle conjugates, our unpublished data showed that the conjugate remained stable beyond 6 months when kept at 4 oC in liquid form.- In available IgG-based LFIAs for fumonisin detection, detection conjugates are dried on conjugate pad, therefore, it is difficult to compare the stability of the developed test with known IgG-based LFIAs. ReferencesLing, S., Pang, J., Yu, J., Wang, R., Liu, L., Ma, Y., et al. (2014). Preparation and identification of monoclonal antibody against fumonisin B(1) and development of detection by Ic-ELISA. Toxicon, 80, 64-72. doi:10.1016/j.toxicon.2013.12.008Maragos, C. M., Sieve, K. K., & Busman, M. (2018). Development of antibodies for N-(1-deoxy-D-fructos-1-yl) fumonisin B1 and cross-reaction with modified fumonisins. World Mycotoxin Journal, 11(4), 493-502. doi:10.3920/WMJ2018.2308Shimizu, M., Nagashima, H., Sano, K., Hashimoto, K., Ozeki, M., Tsuda, K., et al. (1992). Molecular Stability of Chicken and Rabbit Immunoglobulin G. Bioscience, Biotechnology, and Biochemistry, 56(2), 270-274. doi:10.1271/bbb.56.270Yu, F.-Y., & Chu, F. S. (1999). Production and Characterization of Monoclonal Antibodies against Fumonisin B1. Food and Agricultural Immunology, 11(4), 297-306. doi:10.1080/09540109999681Zhang, X., Wang, Z., Fang, Y., Sun, R., Cao, T., Paudyal, N., et al. (2018). Antibody Microarray Immunoassay for Simultaneous Quantification of Multiple Mycotoxins in Corn Samples. Toxins, 10(10), 415. doi:10.3390/toxins10100415" } ] } ]	1	https://f1000research.com/articles/8-1042
https://f1000research.com/articles/8-1508/v1	27 Aug 19	{ "type": "Research Article", "title": "EEG anticipation of random high and low arousal faces and sounds", "authors": [ "Gian Marco Duma", "Giovanni Mento", "Luca Semenzato", "Patrizio E. Tressoldi", "Gian Marco Duma", "Giovanni Mento", "Luca Semenzato" ], "abstract": "Background: In this study, we investigated the neural correlates of the anticipatory activity of randomly presented faces and sounds of both high and low arousal level by recording EEG activity with a high spatial resolution EEG system. Methods: We preregistered the following three hypotheses: 1) a contingent Negative Variation (CNV) difference in the amplitude voltage between auditory vs faces stimuli; 2) a greater amplitude voltage in the CNV, in high arousal stimuli vs low arousal stimuli, both in auditory and faces stimuli, in the temporal window from 0 to 1000 ms before the stimulus presentation; 3) in the time window from 0 to 1000 ms a sensory specific activation at the brain source level in the temporal lobe and auditory cortex before the presentation of an auditory stimulus and an activation of occipital area, dedicated to the elaboration of visual stimuli, before the presentation of faces . Results: Using a preregistered, hypothesis-driven approach, we found no statistically significant differences in the CNV due to an overly conservative correction for multiple comparisons for the control of Type I error. By contrast, using a data-driven approach based on a machine learning algorithm (Support Vector Machine), we found a significantly larger amplitude in the occipital cluster of electrodes before the presentation of faces with respect to sounds, along with a larger amplitude in the right auditory cortex before the presentation of sounds with respect to faces. Furthermore, we found greater CNV activity in the late prestimulus interval for high vs. low-arousal sounds stimuli in the left centro-posterior scalp regions. Conclusions: These findings, although preliminary, seem to support the hypothesis that the neurophysiological anticipatory activity of random events is specifically driven by either the sensory characteristics or the arousal level of future stimuli.", "keywords": [ "anticipation", "random events", "EEG", "source analysis", "support vector machine" ], "content": "Introduction\n\nWhen we observe the world, reality is not always chaotic and unpredictable. Rather, it shows some spatiotemporal regularities. The capacity to extract these regularities is a fundamental survival function for an organism, since it allows for the fine-grained, proactive control of resource allocation necessary for stimuli elaboration or action preparation. Taking advantage of these regularities, the brain building up inner models of external reality anticipating forthcoming events implementing its predictive aptitude by capitalizing on Bayesian computational architecture and, consequently, optimizing our behaviour over time (Friston, 2005; Mento, 2017; Mento & Vallesi, 2016; Mento et al., 2015),\n\nThis account posits the brain as a mechanism that makes continuous inferences about forthcoming stimuli on the basis of conditional probabilistic computations (Chater et al., 2006; Pouget et al., 2013) accomplished by exploiting the sensory contingencies provided by the external sensory environment and the internal representation of the events.\n\nIn the last decade, neuroimaging evidence has shown that the possibility of predicting the ‘what’, ‘when’, and ‘where’ of forthcoming events translates into anticipatory neural activity (Mento et al., 2015; Mento et al., 2018; Mento, 2017; Mento et al., 2013). Notably, the experimental paradigms of this literature took advantage of statistically predictable stimuli, where predictive information about stimulus identity and time–space features were provided by cue presentation. A consistent finding reported in the literature is the observation of a sustained, slow event-related activity preceding the onset of predicted stimuli. This wave, known in literature as the Contingent Negative Variation (CNV; Rebert & Tecce, 1973; Walter et al., 1964), has been interpreted as a general neural marker of anticipatory processes, including motor preparation, temporal expectancy, and temporal attention. Interestingly, the CNV is sensitive to the subjective (rather than objective) implementation of anticipatory processes (Mento et al., 2013; Trillenberg et al., 2000) in the context of temporally predictable events (Mento et al., 2015; Mento, 2017; Miniussi et al., 1999).\n\nRecent studies explored the possibility of extending the study of anticipatory brain activity to statistically unpredictable stimuli (Duggan & Tressoldi, 2018; Duma et al., 2017; Mossbridge et al., 2012; Radin et al., 2011), more generally defined by Mossbridge et al. (2014) as Predictive Anticipatory Activity (PPA). In our previous work (Duma et al., 2017), we investigated brain PPA phenomena in relation to the passive presentation of randomly presented car accidents compared with safe journeys. The results showed a prestimulus greater CNV negativity amplitude for trials related to car crashes from those ending with no car accidents. These results suggested that statistically unpredictable events may nevertheless engage differential anticipatory neural activity. However, since the experimental design we employed in our previous study had possible methodological limitations in the implementation of a pseudo- rather than true-random trial sorting procedure, as well as in the use of a variable anticipatory time window, we designed a new paradigm to further address any possible procedural issues.\n\nTo address any possible methodological pitfalls, in the present work we used a trial randomization procedure mediated by a true random number generator. Unlike the implementation of a pseudo-random trial sorting procedure, this method ensures that the experimental conditions presented to participants are fully randomized, so that any possible bias arising from employing a seed-based trial selection can occur, including casual repetition of the same condition in many consecutive trials. Indeed, this issue could result in the presence of possible biases conditioning both participants’ behavioural and neural correlates. Furthermore, to avoid any possible bias due to implicit temporal expectancy of stimuli induced by a variable prestimulus interval (i.e., hazard function (Mento et al., 2015)), we purposely used a fixed prestimulus time window. As a second goal of the present study, we aimed at extending the knowledge about the nature of PPA phenomena, with the specific interest of investigating whether such phenomena are aspecific (i.e., independent from stimuli and task) or specific (i.e., different from stimulus type and task). To this end, in the current study, a group of healthy volunteers were asked to predict either the sensory category (i.e., visual or auditory) or the emotional meaning (i.e., low or high arousal level) of randomly delivered target stimuli in the context of both a passive and an active experimental task. Finally, in line with the methodological guidelines for increasing the reproducibility of scientific findings, the methodological procedure of this study was pre-registered.\n\n\nMethods\n\nThe method of this study was registered before data collection and is available here: https://osf.io/uf59a\n\nThirty healthy participants recruited from graduate and undergraduate students of Padova University, with normal or corrected-to-normal vision participated in the experiment. Due to the high level of noise artefact, data from two participants were removed from the analysis. All participants received 10€ for their participation in the study. The study was reviewed and approved by the ethical committee of the School of Psychology of the University of Padua (Protocol No. 2278). Before the study, participants signed an informed consent in accordance with the principles expressed in the Declaration of Helsinki.\n\nThe sample size of this study was reduced with respect to what was declared in the pre-registration due to laboratory use limitation. For this reason, we were only able to collect data from 30 participants instead of the 36 declared. This reduction clearly impacted the statistical power of our planned data analyses.\n\nIn the present experiment, we used two sensory categories of stimuli (i.e., visual and auditory), which were extracted from two standardized international archives. Visual stimuli consisted of pictures of 28 faces extracted from the NIMSTIM archive (Tottenham et al., 2009), whereas auditory stimuli consisted of 28 sounds chosen from the International Affective Digitized Sounds (IADS) archive (Stevenson & James, 2008). For each sensory category, the stimuli were further extracted according to their arousal value. We selected 14 neutral faces and 14 fearful faces from the NIMSTIM inventory, and 14 low- and 14 high-arousal sounds were selected from the IADS repertoire and balanced by arousal with the NIMSTIM stimuli set. These materials are available here: https://doi.org/10.6084/m9.figshare.6874871.v3 (Tressoldi et al., 2018).\n\nExperimental paradigm. All participants were presented with two different experimental tasks, which were delivered in separate blocks (see Figure 1). The first was a passive and the second was an active task preceded by a warm-up condition where the type of stimuli was anticipated in the cue signal. Both the block presentation order and the response button were counterbalanced between subjects to avoid possible response biases. The two tasks are described in the Figure 1.\n\nThe figure illustrates the sequence of events and the temporal trial structure relative to the passive (top) and the active (bottom) tasks, which were delivered in blocks. Within each task, the stimuli were randomly presented and equally distributed according to either sensory category (faces or sounds) and arousal level (high or low).\n\nPassive task. As shown in Figure 1, at the beginning of each trial, participants were presented with a warning signal, a fixation cross presented centrally on the screen for 300 ms. After that, a fixed 1000ms blank inter-stimulus interval (ISI) was delivered, followed by a 500ms target stimulus. The target stimulus could be either the picture of a face presented on the centre of the screen or a sound delivered bilaterally through two loudspeakers, with a 50% distribution. Half of the stimuli within each category were low-arousal and the other half were high-arousal, equally distributed. Participants were told that they had to guess which kind of stimulus they would be presented with. No behavioural responses were required until they actually received the stimulus target. At target onset, participants had to discriminate between visual or auditory stimuli by pressing two different buttons on the response box. The response buttons were counterbalanced across participants. After the response, the stimulus target disappeared and a blank screen was presented for a jittered duration between 1000 and 1200 ms (inter-trial interval) before the beginning of the next trial.\n\nActive task. In the active task, event sequence and timing were the same as those in the passive task. As illustrated in Figure 1, the only difference in comparison with the passive task was that, after the prestimulus ISI, participants were presented with a slide showing a central question mark. They were then asked to make an explicit choice about the sensory category of the upcoming stimulus by pressing the response box. This allowed us to obtain an overt behavioural measure of the anticipation of random events as the percentage of correct responses compared to chance-level for each stimulus category. Immediately after participants’ response, stimuli were presented for 500 ms. As with the passive task, the response buttons were counterbalanced across participants.\n\nA total of 200 trials sorted by stimulus category and arousal was presented in each task, for an experiment duration of about 18 minutes. In both tasks, stimuli presentation was fully randomized. Specifically, the trial-type randomization was generated online during the ISI by using a true random number generator (TrueRNG-2™). The TrueRNG hardware uses the avalanche effect in a semiconductor junction to generate true random numbers. Randomization via an external TrueRNG device does not rely on seed-based randomization algorithms, but on current fluctuations within the device, assuring a true random distribution. The RNG was interfaced with the stimuli presentation software E-Prime™ 2.0.8.90.\n\nDuring the entire experiment, the EEG signal was continuously recorded using a Geodesic high-density EEG system (EGI GES-300) through a pre-cabled 128-channel HydroCel Geodesic Sensor Net (HCGSN-128) and referenced to the vertex. Scalp voltages were recorded and amplified during the entire experiment. The sampling rate was 500 Hz. The impedance was kept below 60 kΩ for each sensor. To reduce the presence of EOG artefacts, subjects were instructed to limit both eye blinks and eye movements as much as possible.\n\nFurther detail about analysis, including removal of artefacts, is available from the preregistration record: https://osf.io/uf59a.\n\nEvent-related potential (ERP) analysis. The ERP analysis was performed with Matlab toolbox EEGLAB (Delorme & Makeig, 2004). The EEG was segmented offline starting from 200 ms before the cue onset and ending 300 ms after the stimulus onset. The length of the analysed epoch was 1600 ms, starting from cue onset and included 300 ms of cue/fixation cross presentation, 1000 ms of ISI, and 300 ms from stimulus onset. Our original hypothesis, driven by the results of our previous study (Duma et al., 2017), was that PPA phenomena may rely on a prestimulus sustained ERP activity, presumably resulting in the amplitude modulation of the CNV component. For this reason, our confirmatory, pre-planned analysis purposely targeted the temporal window between 300 ms and 1300 ms from trial onset.\n\nAll epochs were visually inspected to remove bad channels and rare artefacts. Artefact-reduced data were then subjected to Independent Component Analysis (Stone, 2002). All independent components were visually inspected, and those related to eye blinks, eye movements, and muscle artefacts according to their morphology and scalp distribution were discarded. The remaining components were then projected back to the electrode space to obtain cleaner EEG epochs. The remaining epochs containing excessive noise or drift (±100 μV at any electrode) were rejected. After this step, removed bad channels were reconstructed. Data were then re-referenced to the average of all electrodes, and the signal was aligned to the baseline by subtracting the mean signal amplitude in the pre-stimulus interval. Subject average and grand average ERPs were generated for each electrode site and experimental condition. To provide a spatial representation of the anticipatory ERP activity, we used the Brainstorm software’s dedicated software (Tadel et al., 2011). This allowed us to extract videos of obtained time-resolved scalp map projections of the grand average ERP activity from 0 ms to 1350ms.\n\nBrain source analysis. Baseline corrected epochs (-200 to 0 ms) were imported to Brainstorm software (Tadel et al., 2011) with the purpose of reconstructing the cortical generators of pre-stimulus ERP activity. Conductive head volume was modelled according to the Boundary Element Method (BEM) using Open MEEG toolbox (Gramfort et al., 2010; Kybic et al., 2005). The solution space was constrained to the cerebral cortex, which was modelled as a three-dimensional grid of 15002 vertices. Furthermore, the inverse transformation was applied to the Montreal Neurological Institute (MNI) canonical mesh (http://brainmap.org/training/BrettTransform.html) of the cortex to approximate real anatomy. The EEG sensor positions were co-registered with the default anatomical mesh by employing rigid rotations and translations of digitized landmarks (anterior and posterior commissure, interhemispheric scissure, nasion, and left and right tragus). The inverse modelling was based on sLORETA implemented as a routine of the Brainstorm platform. A noise covariance matrix was generated for each participant based on the average baseline time window. For each participant, the sources were projected using the standard anatomical template provided by the MNI, and their activity was transformed in Z scores relative to the baseline.\n\nERP statistical analysis. We compared face vs. sound pre-stimulus ERP activity (300-1300ms) at the sensor level, including all 128 electrodes. Moreover, to further address the role of arousal in anticipation of random events, we also compared the high- and low-arousal stimuli within each sensory category. Given that a 128-channel recording system and a time resolution of 2ms (500 Hz) were employed, performing a systematic comparison of all samples present a severe risk of false positives. Hence, we used a non-parametric, mass-univariate statistical approach based on paired t-test permutations (Groppe et al., 2011) already employed by our group in previous ERP studies (Duma et al., 2017; Mento et al., 2018; Mento, 2017; Mento & Vallesi, 2016; Mento & Valenza, 2016). Specifically, 1000 Monte Carlo permutations with cluster correction were applied, using Fieldtrip (Oostenveld et al., 2011) functions, implemented in Brainstorm.\n\nExploratory analysis. The above-mentioned statistical analyses were preregistered and hypothesis driven. We expected to find that the presence of anticipatory effects of random stimuli was reflected in an amplitude change of the CNV component elicited during the ISI. However, it should be noted that the real nature of anticipatory brain activity of statistically unpredictable stimuli has not been fully explored. Hence, it is possible that the exact spatial and temporal locus of PPA effects could be outside the time windows expected based on previous evidence. Thus, while the use of a confirmatory approach increases the reliability and reproducibility of the results, it may exclude possible interesting effects extending over temporal windows or spatial areas originally ignored in the pre-declared analyses. For this reason, we implemented additional exploratory analyses extending the epoch of interest to the entire pre-stimulus time window (i.e., from the onset of the fixation cross to the end of stimulus presentation) with the aim of looking for possible earlier effects than those expected. To test for any data-driven difference between experimental conditions, a Support Vector Machine (SVM) analysis was performed. The SVM belongs to the class of supervised machine learning models. Given a set of training data, in which the two categories are labelled, the SVM algorithm generates a model that is able to assign new examples to one category or the other. The K-folds cross-validation technique implemented in Brainstorm (Tadel et al., 2011) has been used to validate the SVM model using the ERP of each subject as data. Cross-validation is a resampling procedure for model selection. K-fold approach ‘involves randomly dividing the set of observations into k groups, or folds, of approximately equal size. The first fold is treated as a validation set, and the method is fit on the remaining k − 1 folds’ (James et al., 2013). For each task, we contrasted face and sound stimuli and high- and low-arousal stimuli separately within each stimulus category. The results of the SVM approach are expressed as the dynamic, time-resolved evolution of the discrimination accuracy between two categories. It worth mentioning that Machine Learning (ML) approach with EEG signal shows different results based one the features of the EEG signal to classify, the feature selection criterion and the classifier algorithm (Lotte et al., 2007). Moreover, to the best of our knowledge, ML approach have never been applied to classify brain activity for statistical unpredictable stimulus presentation. For these reasons, we tested different cut-off frequency (20Hz, 10Hz, 7Hz, 5Hz, 4Hz, and 3Hz) in order to understand which frequency maximized the classification accuracy of the algorithm.\n\n\nResults\n\nThe descriptive statistics relative to the percentages of the correct predictions of faces and sounds in the active block are reported in Table 1.\n\nThe inferential statistics based on a one-tailed, one-sample t-test with the null hypothesis of 50% corresponding to the chance level yielded the following results: Faces, t-test = 2.3; p = .01; effect size d = .45; 95% CI = .019 - .85; Sounds, t-test = .97; p = .16; effect size d = .19; 95% CI = -.20 - .60.\n\nThe mean number of artefact-free trials per subject accepted for averaging was comparable both in the passive (Faces: 90 ± 7.79; Sounds: 92 ± 9.3) as well as in the active (Faces: 90.60 ± 10.1; Sounds: 99 ± 10) task. Likewise, the number of high- and low-arousal stimuli within each stimulus category was comparable both in the passive (Low Arousal Faces: 46.6 ± 6.34; High Arousal Faces: 43.4 ± 4.52; Low Arousal Sounds: 46.5 ± 6.6; High Arousal Sounds: 45.67 ± 6.83) and active task (Low Arousal Faces: 46.21 ± 7.6; High Arousal Faces: 44.4 ± 6.8, Low Arousal Sounds: 45.35 ± 7.57; High Arousal Sounds: 43.68 ± 6.56). This confirmed that the true-random trial sorting procedure was effective in generating an equivalent number of trials per condition, ensuring a comparable ERP signal-to-noise ratio.\n\nAs expected based on previous literature (Mento et al., 2013; Walter et al., 1964), the visual inspection of ERP waveforms revealed the presence of a reliable CNV component in all tasks. This component was represented as an increase in fronto-central negativity about 500 ms from trial onset and lasting for the full duration of the ISI duration until stimulus onset. The topographical distribution of this activity for each task is shown in Figure 2 and Figure 3 for face vs. sound and high- vs. low-arousal contrasts, respectively (see also https://doi.org/10.6084/m9.figshare.6874871.v3 (Tressoldi et al., 2018) for a time-resolved dynamic spatial representation of the CNV over the whole scalp).\n\nThe visual inspection of the high scalp-resolution maps obtained by averaging the CNV time window (i.e., 300–1300ms) showed prestimulus differences in the topographical distribution between faces and sounds, with a greater fronto-central negativity for face type stimuli (Figure 2). This difference was less evident in the active block.\n\nConcerning arousal level, the scalp distribution maps displayed an increase in the fronto-central negativity for high-arousal face stimuli compared to low-arousal stimuli in the passive task, as well as a differential map localization of the CNV between high- and low-arousal sound stimuli. Notably, this brain activity pattern was not consistent across tasks. In the active task, low- and high- arousal stimuli elicited a comparable fronto-central negativity, although a larger posterior positivity was evident for low-arousal face and high-arousal sound (Figure 3).\n\nAlthough the visual inspection of the spatial maps revealed qualitative topographical differences, in none of the two tasks did the non-parametric, permutation analyses performed in the pre-registered anticipatory epoch (300–1300ms from warning onset) reveal statistically significant effects, whether when comparing stimulus categories or when contrasting stimuli arousal within categories.\n\nA possible explanation of the lack of significant statistical corroboration of visual differences may be that mass univariate statistical tests are strongly sensitive to the presence of random factors, as in the case of including the whole spatial information (128 electrodes). This may ultimately result in excluding experimental effects strictly localized in specific scalp regions or temporal windows. As mentioned above, the hypotheses about the spatio-temporal properties of PPA induced by the present experimental manipulations were driven by our previous study (Duma et al., 2017), which showed a broad fronto-central and long lasting CNV modulation in anticipation of random simulated car accidents. However, in that study, we used a low spatial resolution EEG system (32 vs. 128 channels) as well as a lower signal acquisition sampling rate (i.e., 250 vs. 500 Hz). In hindsight, this methodological difference may explain the disappearance of significant results due to a strong multiple-comparison correction. In line with this possible explanation, we observed that the fronto-central electrode activity in both tasks showed significant modulatory effects when permutations were performed without applying cluster-based correction for controlling Type 1 error.\n\nOur pre-registered analyses included the reconstruction of the source level maps. Although the massive cluster-based correction for multiple comparisons did not yield statistically significant observed differences between the conditions, we report the cortex activation results in Figure 4.\n\nFrom the cortical source map reconstruction, it is possible to identify a common activation for both tasks and stimulus categories in the superior frontal gyrus bilaterally, namely the Supplementary Motor Area that in literature has been identified as one of the most reliable neural generators of CNV (Mento et al., 2013; Mento et al., 2015; Mento, 2017). In addition to this, the cortical maps suggest the possible presence of stimulus-driven differences in primary sensory areas. In spite of this, the source-level results can be considered merely descriptive, rather than offering reliable hints for any inferential reasoning. In fact, considering the absence of statistically significant results at the sensor level, any strong interpretation from source space findings is to be discouraged as a precaution. While bearing these methodological issues in mind, one should nonetheless take into account that both sensor- and source-level descriptive results could be deemed suggestive of the possible presence of subtle effects to be further explored with ad-hoc analyses. To follow up on this indication, we performed additional exploratory statistical analyses extending over earlier temporal windows in an attempt to specifically focus on the possible presence of specific, fine-grained experimental effects.\n\nSupport vector machine results. The data were analyzed with SVM algorithm by applying different exploratory frequency thresholds (20Hz, 10Hz, 7Hz, 5Hz, 4Hz, and 3Hz, with a folding parameter of 5). The best algorithm performance in terms of experimental condition discrimination was obtained by applying a 4 Hz threshold, which was then set as the target SVM frequency. As shown in Figure 6, the application of SVM in the passive task succeeded in discriminating between Face and Sound in the time window 100–300ms, reaching a discrimination accuracy value of 71.43% (see Figure 5).\n\nIn the active task, the SVM was able to correctly differentiate between high- and low-arousal sounds, reaching a decoding accuracy of 68%, but in a later temporal window, between 1100 and 1300ms (see Figure 6).\n\nTo statistically confirm the SVM findings while accounting for all spatial information, we applied cluster-based permutation statistics taking into account all 128 channels, this time targeting the specific contrasts and temporal windows indicated by the SVM algorithm as exhibiting effects.\n\nConcerning the passive task, the results showed a bilateral posterior significant cluster of electrodes (p = .01966, cluster statistic = 39, cluster size = 28) when we compared face and sound stimuli in the 100–300ms time window. Concerning the active task, we found a significant left centro-posterior cluster (p = .047, cluster statistic = 25, cluster size = 18) when high-arousal sounds are compared to low-arousal sounds. These differences are presented in Figure 7.\n\na) in the occipital cluster for face vs sound stimuli comparison in the passive task; b) in the parietal cluster for high- vs low-arousal sound comparison in the active task.\n\nTo further address the functional nature of these phenomena, we performed the reconstruction of the cortical source of both effects described above. Both the source-level topological distribution (source mean activity between 300–1300ms) and temporal evolution over the whole prestimulus epoch are depicted in Figure 8.\n\nThe time series below represent the time evolution of the source activation, highlighting the difference between 100–300ms (blue rectangle), respectively of the lateral occipital left (on the left) and superior temporal right (on the right).\n\nRelative to the early anticipatory effect, we observed a higher recruitment of the occipital–parietal left cortical regions, especially the lateral occipital gyrus, which was more activated before the presentation of faces regardless of the arousal they expressed. The visual inspection of the temporal dynamics showed that this region is highly activated at about 200 ms from the presentation of the fixation cross when a face was presented. No other relevant modulations are present in the late prestimulus interval spanning the CNV timing. A second remarkable finding was a larger recruitment of neural activity in the right superior temporal gyrus, again at about 200ms when a sound was presented. Both of the above findings corroborate our original, pre-declared hypothesis that PPA brain activity is category-dependent, since it engages in the same primary sensory areas that will be activated after target presentation. In other words, the anticipation of a face pre-activates visual occipital areas lefts lateralized, whereas the anticipation of a sound pre-activates auditory temporal areas more lateralized in the right hemisphere.\n\nWhen performed on the second temporal window showing late prestimulus ERP amplitude modulation between high- and low-arousal auditory stimuli in the active task, the cortical reconstruction showed that high-arousal stimuli were preceded by the recruitment of a broader cortical network as compared to low-arousal stimuli. Indeed, in both cases, the fronto-central areas around the SMA were commonly activated. However, when the fixation was actually followed by the presentation of high-arousal stimuli, we observed an additional involvement of the left superior parietal area, maximally expressed between 1100 and 1300ms from its onset (see Figure 9).\n\nBelow in the figure, it is represented the time course of the superior parietal left area of the Desikan-Killaney atlas.\n\n\nDiscussion\n\nIn a recent study (Duma et al., 2017), we reported evidence of differential anticipatory brain activity preceding unpredictable, simulated car crashes or safe journeys. Here, we sought to further investigate the evidence of psychophysiological predictive activity (PPA; Mossbridge et al., 2014) of random events while further ruling out potential methodological shortcomings due to the randomization process and stimulus timing. As a general consideration, it must be taken into account that the available evidence in existing literature about the neurophysiological mechanisms underpinning the anticipation of random events is limited. Hence, it is difficult to generate clear and reliable hypotheses with regard to the spatial and temporal neural locus of such phenomena. Notwithstanding this caveat, a commonly accepted idea is that if the brain is able to anticipate statistically unpredictable random stimuli, different forthcoming stimuli categories should rely on specific and dissociable anticipatory neural patterns. More precisely, this anticipatory effect might be reflected in the pre-activation of the brain regions that subtend the elaboration of the physical features of upcoming stimuli (e.g., a pre-activation of the auditory cortical regions preceding the onset of a sound as well as of visual regions preceding face presentation). Following this line of reasoning in our previous work, we had argued that the capability of anticipating potentially threatening events (e.g. simulated car accidents) may result in the mobilization of psychophysical resources to implement a possible reaction, this mechanism putatively playing a key role in survival.\n\nBased on the above assumptions, the aim of the present study was two-fold. On the one hand, we tried to address some potential methodological issues that may have somehow biased our previous study. These included either the implementation of a pseudo-random instead of truly random trial sorting procedure or the use of a variable temporal trial structure. On the other hand, we tried to extend our previous results into other stimuli category with the aim of deepening the understanding of the sources of anticipatory brain activity of randomly presented stimuli.\n\nTo address these goals in the present study, we recorded the high spatial resolution EEG activity when presenting two different stimulus categories in a random, unpredictable sequential order. These included pictures of faces and sounds as visual and auditory sensory categories, respectively. Moreover, we further manipulated the psychophysiological activation meaning of those categories by matching them by arousal level (high or low). All stimuli were randomly presented by interfacing with a true random number generator. As in our original hypothesis, we expected to identify at the EEG sensor level a stimulus type and/or arousal level dependent difference during the prestimulus time window. Specifically, following our pre-registered hypothesis we expected to observe some stimulus-driven amplitude differential effects arising from about 1000ms before stimulus onset. Moreover, we hypothesized that such differences may stem from specific neural patterns mimicking the sensory nature of the stimulus. That is, we expected to observe source-level pre-activation of visual cortical areas before faces and pre-activation of auditory cortical areas before sounds.\n\nThe cluster-based permutation analyses of the target pre-stimulus time window (i.e., 300–1300ms from trial onset) showed only a marginally significant CNV modulation due to experimental manipulation. Indeed, the application of a massive cluster-based multiple-comparison correction taking into account the whole spatial information (128 electrodes) as well as a huge temporal window (as long as 1 second, i.e., between 300 and 1300ms from trial onset) did not confirm this effect from a statistically point of view.\n\nAs mentioned above, a possible explanation for this null effect is that the implementation of a whole-scalp analysis may have been too conservative for a subtle experimental effects. In fact, in our previous study (Duma et al., 2017) we used a lower resolution EEG system (32 electrodes), which ultimately resulted in a considerably smaller number of multiple comparisons. In other words, the analysis of a large, complex spatiotemporal dataset may have requested a stronger statistical power.\n\nHowever, we decided to further analyse our data by applying an exploratory approach. To this purpose we used an SVM as a machine-learning classificatory algorithm. In the passive task, this analysis revealed a data-driven discrimination accuracy of 71.43% between visual and auditory stimuli which occurred in an early temporal window (100–300ms from trial onset). In the active task, the SVM algorithm revealed a later data-driven effect (maximum peak 68%) spanning in the late CNV range (1100–1300ms from trial onset), which was specific for the arousal level. It should be underlined that SVM results have been used as a guide indicating in which conditions and temporal windows focusing the analysis. These results were further confirmed by a cluster-based permutation statistics, which were performed targeting the specific temporal windows identified by the SVM. In fact, by applying cluster-based permutation statistics over the temporal window (100–300ms) and conditions (passive face vs. sound), as evidenced by SVM results, we observed a significant bilateral occipital cluster of electrodes showing larger amplitude when the fixation cross at trial onset was followed by visual than auditory stimuli. These prestimulus activities were supported by different cortical networks. Indeed, we found that the visual areas (i.e., the left lateral occipital lobe) were more activated at about 200ms from trial onset in trials displaying faces than in those delivering sounds. Differently the right auditory cortex was more activated in trials containing sounds than in those delivering faces.\n\nAdditionally, we found that in the active task when participants were explicitly asked to guess the category of the forthcoming stimulus (face or sound) the percentage of correct responses (52%) was significantly higher than chance level. This behavioural finding was accompanied by a larger CNV activity in the late prestimulus interval (1100–1300ms) for high- vs. low-arousal stimuli. This effect was localized in the left centro-posterior scalp regions and originated from a specific pre-activation of the left superior parietal cortex, which showed a larger sustained activity before high-arousal stimuli in the late prestimulus interval. Although the exact functional role of the superior parietal cortex in the anticipation of random, high-arousal sounds is not well understood on the basis of current scanty literature, a possible generic explanation may maintain that this region is involved in the pre-allocation of cognitive resources needed to process predicted high-arousal stimuli.\n\nAlthough obtained by exploratory, rather than originally planned, confirmatory hypotheses, these results are in line with the expectations of a stimulus-dependent pre-activation of those brain regions that will be engaged in processing the upcoming stimulus.\n\nWhile we feel reasonably confident in excluding that the presence of anticipatory effects of random events may have been biased by possible methodological shortcomings, the interpretation of the possible nature of such phenomena is still debated. In line with one of the most shared theoretical accounts, our results may be explained in the theoretical framework of the Predictive Anticipatory Activity (PAA) of Mossbridge et al. (2014). The underlying assumption of this kind of empirical evidence maintains that PAA might be thought of as an unconscious anticipation of a conscious future events, suggesting the possibility of quantum-like temporal entanglement effects in human physiological processes (Fisher, 2015; Hameroff, 2012; Jedlicka, 2017; Tressoldi et al., 2015). The endorsement of quantum-like phenomena as possible mediating mechanisms of physiological processes taking place in random event prediction, implies the possibility of temporal symmetry in the interpretation of reality (Reznik & Aharonov, 1995) or even possible retrocausal effects (Ma et al., 2012).\n\nWhatever the specific physical and physiological nature of anticipatory mechanisms, which is beyond the original purpose of the present study, we report a methodologically-controlled and replicable experimental setting that suggests the reliability of PPA phenomena. To the best of our knowledge, this is the first work in which advanced techniques, such as high-spatial resolution EEG and source reconstruction, have been applied to the investigation of PPA phenomena in combination with the implementation of both confirmatory and exploratory methodological approaches. This allowed us to present new data complementing previous evidence because we argue that PPA phenomena may be driven by specific brain processes, which involves discrete brain areas according to the sensorial category of the upcoming stimulus.\n\nIn scientific literature, criticism have been raised regarding the anticipatory effects for statistically unpredictable stimuli (Galak et al., 2012; Ritchie et al., 2012; Wagenmakers et al., 2011), criticizing the findings reported by Bem (2011). Nonetheless, we believe that those criticisms cannot be related to our work, both for theoretical and methodological reasons. First, Bem’s results were referred to the so called ‘Retroactive Facilitation of Recall’. In fact, as stated by the author, in his experimental task “Participants were first shown a set of words and given a free recall test of those words. They were then given a set of practice exercises on a randomly selected subset of those words. The psi hypothesis was that the practice exercises would retroactively facilitate the recall of those words, and, hence, participants would recall more of the to-be-practiced words than the unpracticed words” (Bem, 2011). By contrast, our theoretical framework emerges from the already published results of Radin et al. (2011), Mossbridge et al. (2012), Mossbridge et al. (2014), and Duma et al., 2017, who investigated physiological activity (EEG, EMG, pupillary response) before stimuli randomly presented, finding positive results. Therefore, considering the evidence already published, and the evident difference between Bem’s study and the one we are presenting, both in the type of measures (behavioural vs. electrophysiological), and the experimental paradigm (words recall vs. face/sound passive presentation), we believe that the possible criticisms raised by the abovementioned authors cannot be by default applied to all the studies investigating anticipatory phenomena, including the present one.\n\nWe are aware that the present topic needs additional confirms from scientific literature. For this reason, we believe that the present results may be a stimulus to other researchers to investigate, with a rigorous and fully reproducible methodological approach, the real nature of anticipatory effects for statistically unpredictable stimuli, deepening in this way our understanding.\n\nFor example, future replications of the present study may include single-subject MRI scans with the objective of localizing a single subject’s visual and auditory cortex, using those areas as ROI to be entered in a multivariate statistic approach. Taking in account the growing body of literature providing experimental results to the human capability of anticipating randomly presented stimuli, we believe that this topic deserves a precise and rigorous scientific investigation, as it happens for all cognitive human activities, in order to understand this controversial but nevertheless fascinating phenomena.\n\n\nData availability\n\nFigshare: EEG anticipation of random high and low arousal faces and sounds, https://doi.org/10.6084/m9.figshare.6874871.v4 (Tressoldi et al., 2018).\n\nThis project contains that following underlying data:\n\nEEG metafile;\n\nEEG data related to the Passive, Active and Predictive conditions;\n\nHigh (H) and Low (L) arousal visual and auditory stimuli;\n\nVideo clips of the EEG activity before stimulus presentation.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nNIMSTIM archive materials available from http://www.macbrain.org/resources.htm. Registration and acceptance of the terms and conditions of the website must be completed in order to use the stimulus set.\n\nInternational Affective Digitized Sounds (IADS) archive materials available from: https://csea.phhp.ufl.edu/media/iadsmessage.html. Those wishing to use these materials must submit the IADS Researcher Information Form and sign the IADS User Agreement.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBem DJ: Feeling the future: experimental evidence for anomalous retroactive influences on cognition and affect. J Pers Soc Psychol. 2011; 100(3): 407–425. PubMed Abstract \| Publisher Full Text\n\nChater N, Tenenbaum JB, Yuille A: Probabilistic models of cognition: conceptual foundations. Trends Cogn Sci. 2006; 10(7): 287–291. PubMed Abstract \| Publisher Full Text\n\nDelorme A, Makeig S: EEGLAB: an open source toolbox for analysis of single-trial EEG dynamics including independent component analysis. J Neurosci Methods. 2004; 134(1): 9–21. PubMed Abstract \| Publisher Full Text\n\nDuggan M, Tressoldi P: Predictive physiological anticipatory activity preceding seemingly unpredictable stimuli: An update of Mossbridge et al’s meta-analysis [version 2; peer review: 2 approved]. F1000Res. 2018; 7: 407. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDuma GM, Mento G, Manari T, et al.: Driving with Intuition: A Preregistered Study about the EEG Anticipation of Simulated Random Car Accidents. PLoS One. 2017; 12(1): e0170370. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFisher MP: Quantum cognition: the possibility of processing with nuclear spins in the brain. Ann Phys. 2015; 362: 593–602. Publisher Full Text\n\nFriston K: A theory of cortical responses. Philos Trans R Soc Lond B Biol Sci. 2005; 360(1456): 815–836. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGalak J, LeBoeuf RA, Nelson LD, et al.: Correcting the past: failures to replicate ψ. J Pers Soc Psychol. 2012; 103(6): 933–948. PubMed Abstract \| Publisher Full Text\n\nGramfort A, Papadopoulo T, Olivi E, et al.: OpenMEEG: opensource software for quasistatic bioelectromagnetics. Biomed Eng Online. 2010; 9(1): 45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGroppe DM, Urbach TP, Kutas M: Mass univariate analysis of event-related brain potentials/fields I: a critical tutorial review. Psychophysiology. 2011; 48(12): 1711–1725. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHameroff S: How quantum brain biology can rescue conscious free will. Front Integr Neurosci. 2012; 6: 93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJames G, Witten D, Hastie T, et al.: An introduction to statistical learning. New York: springer. 2013; 112: 181. Publisher Full Text\n\nJedlicka P: Revisiting the Quantum Brain Hypothesis: Toward Quantum (Neuro)biology? Front Mol Neurosci. 2017; 10: 366. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKybic J, Clerc M, Faugeras O, et al.: Fast multipole acceleration of the MEG/EEG boundary element method. Phys Med Biol. 2005; 50(19): 4695–710. PubMed Abstract \| Publisher Full Text\n\nLotte F, Congedo M, Lécuyer A, et al.: A review of classification algorithms for EEG-based brain-computer interfaces. J Neural Eng. 2007; 4(2): R1–R13. PubMed Abstract \| Publisher Full Text\n\nMa XS, Zotter S, Kofler J, et al.: Experimental delayed-choice entanglement swapping. Nat Phys. 2012; 8(6): 479–484. Publisher Full Text\n\nMento G: The role of the P3 and CNV components in voluntary and automatic temporal orienting: A high spatial-resolution ERP study. Neuropsychologia. 2017; 107: 31–40. PubMed Abstract \| Publisher Full Text\n\nMento G, Astle DE, Scerif G: Cross-frequency Phase-Amplitude Coupling as a Mechanism for Temporal Orienting of Attention in Childhood. J Cogn Neurosci. 2018; 30(4): 594–602. PubMed Abstract \| Publisher Full Text\n\nMento G, Tarantino V, Sarlo M, et al.: Automatic temporal expectancy: a high-density event-related potential study. PLoS One. 2013; 8(5): e62896. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMento G, Tarantino V, Vallesi A, et al.: Spatiotemporal neurodynamics underlying internally and externally driven temporal prediction: a high spatial resolution ERP study. J Cogn Neurosci. 2015; 27(3): 425–39. PubMed Abstract \| Publisher Full Text\n\nMento G, Valenza E: Spatiotemporal neurodynamics of automatic temporal expectancy in 9-month old infants. Sci Rep. 2016; 6: 36525. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMento G, Vallesi A: Spatiotemporally dissociable neural signatures for generating and updating expectation over time in children: A High Density-ERP study. Dev Cogn Neurosci. 2016; 19: 98–106. PubMed Abstract \| Publisher Full Text\n\nMiniussi C, Wilding EL, Coull JT, et al.: Orienting attention in time. Modulation of brain potentials. Brain. 1999; 122(Pt 8): 1507–18. PubMed Abstract \| Publisher Full Text\n\nMossbridge J, Tressoldi PE, Utts J: Predictive physiological anticipation preceding seemingly unpredictable stimuli: a meta-analysis. Front Psychol. 2012; 3: 390. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMossbridge J, Tressoldi PE, Utts J, et al.: Predicting the unpredictable: critical analysis and practical implications of predictive anticipatory activity. Front Hum Neurosci. 2014; 8: 146. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOostenveld R, Fries P, Maris E, et al.: FieldTrip: Open source software for advanced analysis of MEG, EEG, and invasive electrophysiological data. Comput Intell Neurosci. 2011; 2011: 156869. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPouget A, Beck JM, Ma WJ, et al.: Probabilistic brains: knowns and unknowns. Nat Neurosci. 2013; 16(9): 1170–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRadin DI, Vieten C, Michel L, et al.: Electrocortical activity prior to unpredictable stimuli in meditators and nonmeditators. Explore (NY). 2011; 7(5): 286–299. PubMed Abstract \| Publisher Full Text\n\nRebert CS, Tecce JJ: A summary of CNV and reaction time. Electroencephalogr Clin Neurophysiol. 1973; 33: 173–178.\n\nReznik B, Aharonov Y: Time-symmetric formulation of quantum mechanics. Phys Rev A. 1995; 52(4): 2538–2550. PubMed Abstract \| Publisher Full Text\n\nRitchie SJ, Wiseman R, French CC: Failing the future: three unsuccessful attempts to replicate Bem's 'retroactive facilitation of recall' effect. PLoS One. 2012; 7(3): e33423. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStevenson RA, James TW: Affective auditory stimuli: characterization of the International Affective Digitized Sounds (IADS) by discrete emotional categories. Behav Res Methods. 2008; 40(1): 315–321. PubMed Abstract \| Publisher Full Text\n\nStone JV: Independent component analysis: an introduction. Trends Cogn Sci. 2002; 6(2): 59–64. PubMed Abstract \| Publisher Full Text\n\nTadel F, Baillet S, Mosher JC, et al.: Brainstorm: a user-friendly application for MEG/EEG analysis. Comput Intell Neurosci. 2011; 2011: 879716. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTottenham N, Tanaka JW, Leon AC, et al.: The NimStim set of facial expressions: judgments from untrained research participants. Psychiatry Res. 2009; 168(3): 242–249. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTressoldi P, Duma GM, Mento G: EEG anticipation of random high and low arousal faces and sounds. figshare. Dataset. 2018. http://www.doi.org/10.6084/m9.figshare.6874871.v4\n\nTressoldi PE, Maier MA, Buechner VL, et al.: A macroscopic violation of no-signaling in time inequalities? How to test temporal entanglement with behavioral observables. Front Psychol. 2015; 6: 1061. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrillenberg P, Verleger R, Wascher E, et al.: CNV and temporal uncertainty with 'ageing' and 'non-ageing' S1-S2 intervals. Clin Neurophysiol. 2000; 111(7): 1216–1226. PubMed Abstract \| Publisher Full Text\n\nWagenmakers EJ, Wetzels R, Borsboom D, et al.: Why psychologists must change the way they analyze their data: the case of psi: comment on Bem (2011).2011; 1(7): 1216–1226.\n\nWalter WG, Cooper R, Aldridge VJ, et al.: Contingent Negative Variation: an Electric Sign of Sensorimotor Association and Expectancy in the Human Brain. Nature. 1964; 230: 380–384. PubMed Abstract \| Publisher Full Text" }	[ { "id": "54538", "date": "18 Oct 2019", "name": "J. Bruno Debruille", "expertise": [ "Reviewer Expertise Event-related brain potentials and cognition" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFor this first round of review, the first thing to do is to add the ERPs elicited by the cross from the time the baseline started to be computed up to the end of the ERPs elicited by the stimuli for all experimental conditions.\n\nThe type of electrodes (which material?) and the characteristics of the amps and their band pass have to be included in the method section as well as the gain used. Then, the digital band pass used to process the EEG have to be added (with the details about the cut-offs).\nGiven that the CNV is usually maximal at central or fronto-central electrode sites, data will first have to be re-referenced to one (or two linked) electrodes that are as far as possible from these sites, in order to boost CNV amplitudes and see details. This might also boost effect sizes and give simple statistical analyses a chance to find CNV differences.\nThese ERPs should be in a figure including a sufficient number of channels (e.g, 20) to give a general idea of the scalp distribution of every component. This will allow for a comparison of the CNV activity found with those found in prior CNV studies.\n\nIn this reprocessing, bad channels should not be removed and reconstructed.\nGiven the extremely high scientific stakes of this study, a simple and FULLY reproducible technique should be used as a strat (even ICA is not fully replicable, its results vary a bit). Any fancy technic should be removed first so that readers can have an idea of the ERPs and CNV obtained.\nTo do the reprocessing, I suggest to focus on the 20 channels mentioned above. Each time one channel is bad in an EEG epoch: the whole trial should be suppressed. Please give the trial selection criteria (max voltage, flat line...).\nThen, please mention the average number of trials that had to be rejected for each experimental condition and the standard deviation across participants.\nI will be glad to review the study again and will provide new comments fast according to new results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5059", "date": "12 Dec 2019", "name": "Patrizio Tressoldi", "role": "Author Response", "response": "We are very grateful to dr. Debruille for his insightful comments and suggestions. In the current revision we tried to address, to the best of our possibility, most of the issues he raised. Specifically, we provided additional data and pictures by plotting averaged waveforms from a restricted number of electrodes (n=19) to allow an easy visual inspection of the results. These data have been added as Supplementary Materials available at: https://doi.org/10.6084/m9.figshare.6874871.v5. Moreover, to provide the reviewer with an easy comparison of the waveform pattern with previous CNV literature, we reported a plot of the whole epoch waveforms by using a linked-mastoid reference (see Figure 1b in the Supplementary Materials).Notwithstanding this, we would like to point out that the dataset was not re-analyzed according to what suggested by the reviewer. In the below point-by-point letter we thoroughly explain the rationale of our choice.Reply to the reviewer are provided in italic. For this first round of review, the first thing to do is to add the ERPs elicited by the cross from the time the baseline started to be computed up to the end of the ERPs elicited by the stimuli for all experimental conditions. A: We are happy to provide the ERPs of all the conditions starting from the fixation cross as suggested. To make easy the visual inspection of the waveforms we selected 19 electrodes according to the 10:20 system. The new pictures show the comparison between the visual and auditory conditions separately for the active and the passive task. 2.The type of electrodes (which material?) and the characteristics of the amps and their band pass have to be included in the method section as well as the gain used. Then, the digital band pass used to process the EEG have to be added (with the details about the cut-offs). A: We thank the reviewer for the suggestion, we added the information required in the Method section of the manuscript. 3.Given that the CNV is usually maximal at central or frontocentral electrode sites, data will first have to be rereferenced to one (or two linked) electrodes that are as far as possible from these sites, in order toboost CNV amplitudes and see details. This might also boost effect sizes and give simple statistical analyses a chance to find CNV differences. A: We understand the reviewer’s point and we thank him for the opportunity to better explain the choice of using an average reference. When using high-density EEG system, the average reference is the gold standard for referencing, as explained by Michel, C. M., Koenig, T., Brandeis, D., Gianotti, L. R., & Wackermann, J. (2009). In fact, there is not an electrical inactive point on the scalp, neither the mastoids. However, as stated by these authors “[…] the properties of the EEG forward solution are such that for any source, the voltage integral across the entire head surface is zero. If we could cover the entire head with a sufficient number of electrode, we could approximate this voltage integral by the sum of the measurements at all electrodes. Accordingly, we could assume that the sum of the potential differences from all recorded electrodes would be equal to zero. The sum of the potential differences would thus approximate a ‘’correct’’ zero-reference. Mathematically, this is achieved by using as a reference the average of the measurement at all electrodes. This reference is called, average reference. The validity of the assumption […] depends on the goodness of coverage of the head by the electrode array” (p35, Michel, C. M., Koenig, T., Brandeis, D., Gianotti, L. R., & Wackermann, J. (Eds.). (2009). Electrical neuroimaging. Cambridge University Press). Based on this statement we could assume a that a good coverage of the scalp, as the one provided by an array of 128 geodesic system, would justify the application of the average reference. Moreover, several papers by both our and other groups have been already published with the use of the average reference successfully reporting CNV task-dependent modulations (Mento; Tarantino, Vallesi, Bisiacchi, 2015; Mento and Vallesi, 2016; Mento, 2017; Jang, Jones, Milne, Wilson, Lee, 2016). Furthermore, considering also the statistical approach we used, which includes a whole-scalp explorative analysis, 4.These ERPs should be in a figure including a sufficient number of channels (e.g, 20) to give a general idea of the scalp distribution of every component. This will allow for a comparison of the CNV activity found with those found in prior CNV studies. In this reprocessing, bad channels should not be removed and reconstructed. Given the extremely high scientific stakes of this study, a simple and FULLY reproducible technique should be used as a strat (even ICA is not fully replicable, its results vary a bit). Any fancy technic should be removed first so that readers can have an idea of the ERPs and CNV obtained. To do the reprocessing, I suggest to focus on the 20 channels mentioned above. Each time one channel is bad in an EEG epoch: the whole trial should be suppressed. Please give the trial selection criteria (max voltage, flat line...). Then, please mention the average number of trials that had to be rejected for each experimental condition and the standard deviation across participants. A: We agree that ICA manual components removal may change a bit across experimenters, nonetheless it is a reliable procedure commonly accepted by ERP scientific community, as stated by several studies (Mennes, M., Wouters, H., Vanrumste, B., Lagae, L., & Stiers, P. (2010). Validation of ICA as a tool to remove eye movement artifacts from EEG/ERP. Psychophysiology, 47(6), 1142-1150; Jung, T. P., Humphries, C., Lee, T. W., Makeig, S., McKeown, M. J., Iragui, V., & Sejnowski, T. J. (1998). Extended ICA removes artifacts from electroencephalographic recordings. In Advances in neural information processing systems (pp. 894-900); Li, R., & Principe, J. C. (2006, August). Blinking artifact removal in cognitive EEG data using ICA. In 2006 International Conference of the IEEE Engineering in Medicine and Biology Society (pp. 5273-5276). IEEE. Drisdelle, B. L., Aubin, S., & Jolicoeur, P. (2017). Dealing with ocular artifacts on lateralized ERPs in studies of visual‐spatial attention and memory: ICA correction versus epoch rejection. Psychophysiology, 54(1), 83-99.). Moreover, in our case the variability intrinsic to this procedure has been sensibly reduced since we exclusively targeted eye-, muscle- and heartbeat-related artifacts. As an additional consideration, with the High-Density EEG, the ICA is the best way to correct artifacts specially in the case of eye-related artifacts. In fact, due to the high number of electrodes around the eyes (around 20), their activity is very spread across frontal electrodes, especially if considering that an average reference usually magnifies dipolar scalp configuration. This in turn may result in the contamination of posterior electrodes, that could show residual eye-related artefactual activity overlapped to the CNV. It follows that simply applying a threshold would reject lots of trials, seriously undermining the signal-to-noise ratio and underpowering the statistical comparison. In other words, while we in principle agree with the reviewer that an artifact correction procedure may introduce some variability in the reproducibility of the results, at the same time we believe that a too rigid cleaning procedure based on a threshold-based artifact rejection may 1) not being effective in eye-related artifact cleaning and 2) cause an excessive lack of statistical power due to a dramatic reduction of the signal-to-noise ratio resulting from a massive trial rejection. For this reason, we still believe that ICA is the best approach in the High-Density EEG recording. Anyway, in order to assure replicability, we could provide upon request a list of the components removed for each subject. Concerning bad channels interpolation removal and reconstruction, we would assure the reviewer that this is a standard method of HD-EEG preprocessing, also suggested in the Makoto’s pipeline for EEG analysis and in the Prepipeline toolbox, (https://vislab.github.io/EEG-Clean-Tools/), which is a method to standardize EEG recordings across experimenters. Given that, it is not possible to avoid bad channel identification and correction. We also want to specify that the number of interpolated electrodes was very low (around 8-10 electrodes over 128) and was restricted to the external belt ones surrounding the ears, as a possible aspect of the geodesic sensor net is that those electrodes sometime do not adhere perfectly to the scalp. Concerning the mean number of trials and standard deviation for each experimental condition across participants we added this information in the manuscript, see Table 1. Finally, we would like to add that we are really pleased that our manuscript found the reviewer’s interest. Nonetheless, we desire to underline that the main purpose of the paper is to bring the scientific community attention to the possibility that random anticipatory effect may be actual, in order to generate independent replication of our findings." } ] }, { "id": "54053", "date": "19 Nov 2019", "name": "Xifu Zheng", "expertise": [ "Reviewer Expertise Affective neuroscience" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript \"EEG anticipation of random high and low arousal faces and sounds\" reports results from a study involving anticipatory activity, wherein sensory characteristic and arousal level are experimentally manipulated. By using a machine learning algorithm (Support Vector Machine), they found a significantly larger amplitude in occipital cluster of electrodes before the presentation of faces with respect to sounds, along with a larger amplitude in the right auditory cortex before the presentation of sounds with respect to faces. The study is well designed and well written; the results are interesting and add insight to the ongoing effort to understand the sources of anticipatory brain activity. In addition, the authors adopt open science practices, which increase the level and quality of reporting. There are a few minor issues to address:\nThe authors should report exact number of participants in last paragraph of their introduction, instead of \"a group of healthy volunteers\".\n\nThe sample size is somehow small, and is not consistent with what they declared in the OSF, the reviewer believe 6 more subject will increase the power, and it's not that difficult to have 6 more.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5060", "date": "12 Dec 2019", "name": "Patrizio Tressoldi", "role": "Author Response", "response": "1. The authors should report exact number of participants in last paragraph of their introduction,instead of \"a group of healthy volunteers\".Reply: We thank the reviewer for the suggestion we added this information in the new version of the manuscript.2. The sample size is somehow small, and is not consistent with what they declared in the OSF, thereviewer believe 6 more subject will increase the power, and it's not that difficult to have 6 more.Reply: Unfortunately, in this moment we are not able to increase the sample size due to laboratory and technical issue. However, with respect to the preregistered statistical power, with the final number of participants it reduced from .90 to .82 which is still an acceptable level.Furthermore, we are planning a multicentric study, involving other labs to run a confirmatory study, based on the present results." } ] } ]	1	https://f1000research.com/articles/8-1508
https://f1000research.com/articles/8-2078/v1	10 Dec 19	{ "type": "Systematic Review", "title": "A systematic review and meta-analysis of safety and efficacy of safinamide for motor fluctuations in patients with Parkinson's disease", "authors": [ "Mohamed Abdelalem Aziz Ahmed" ], "abstract": "Background: Safinamide, a recently developed drug with several mechanisms of action has been investigated as an add-on therapy for Parkinson's disease patients suffering from motor complications due to the usage of anti-Parkinson's medications such as levodopa and dopaminergic drugs. The aim of the study is to investigate the efficacy and safety of Safinamide as add-on therapy for Parkinson's disease patients.\n\nMethods: A computerized literature search was conducted of PubMed, EMBASE, ClinicalTrial.gov and Cochrane Library until August 2019. We selected relevant randomized controlled trials comparing safinamide groups to placebo groups. Relevant outcomes were pooled as mean difference (MD) and risk ratio (RR) using Review Manager 5.3. Results: We found that the overall MD of changes in “off-time” and “on time without troublesome dyskinesia” favored the safinamide group over the placebo group (MD -0.72 h, 95% CI -0.89 to -0.56 and MD 0.71 h, 95% CI 0.52 to 0.90, respectively). Additionally, the overall MD of change in Unified Parkinson's Disease Rating Scale part three (UPDRS III) favored the safinamide group (MD -1.83, 95% CI -2.43 to -1.23). In case of adverse events, the pooled meta-analysis did not favor the safinamide group over the placebo group. Conclusions: In this study, we provide class I evidence about the potential role of safinamide as an add-on therapy for Parkinson's disease patients suffering from motor fluctuations. However, a few included studies did not mention the data of important outcomes. Also, we report high risk of bias in individual studies. Future randomized controlled trials with different doses are recommended to provide more evidence for the efficacy and safety of safinamide as a treatment for motor complications of anti-Parkinson's medications.", "keywords": [ "Safinamide", "Parkinson's disease", "Motor", "Dopamine agonist", "Movement disorder", "UPDRS", "Dopamine", "Add-on" ], "content": "Introduction\n\nParkinson’s disease prevalence in the fourth decade of life is 41 people per 100,000 and increases to 1,900 people per 100,000 among those who are older than 801. According to these statistics, Parkinson’s disease is the second most common neurodegenerative disease after Alzheimer’s disease.\n\nThe main pathology of Parkinson’s disease is loss of dopaminergic innervation in the nigrostriatal pathway and spread to various regions in the brain. This loss leads to two types of symptoms; motor and non-motor. Motor symptoms include tremors, rigidity, and bradykinesia. Non-motor symptoms include depression, inability to sustain attention, and sometimes psychosis, especially hallucinations.\n\nDespite the recent medications and updates in the field of pharmacology, there is no definitive treatment that can stop the progress of dopamine receptor loss in the nigrostriatal pathway. Therefore, we only use symptomatic medications for both the motor and non-motor symptoms.\n\nThe main symptomatic medications of Parkinson’s disease are Levodopa (L-Dopa)2,3, dopamine agonists (DAs), and monoamine oxidase-B (MAO-B) inhibitors. Unfortunately, increasing the dose of these medications, especially L-Dopa, leads to motor side effects such as end-of-dose wearing off and dyskinesia, which can be irritating for patients4–6. Recently, a novel drug called safinamide was developed, which can reduce the side effects of these symptomatic medications, especially motor adverse events.\n\nSafinamide has several dopaminergic and non-dopaminergic mechanisms of action such as sodium channel blockade, calcium channel modulation, and MAO-B inhibition7. The main goal of these mechanisms is inhibiting glutamate release and subsequently, improving motor symptoms8.\n\nRecently published studies discussed the beneficial role of safinamide for treatment of motor complications of Parkinson’s medications9–14. Some of them suggest that usage of safinamide improves quality of life and delays the motor deterioration of Parkinson’s disease; thus, our study aims to evaluate the safety and efficacy of safinamide use for Parkinson’s patients. According to our knowledge, this is the first meta-analysis that provides class I evidence for the useful usage of Safinamide for Parkinson’s motor complications.\n\n\nMethods\n\nThe Preferred Reporting Items of Systematic reviews and Meta-Analyses (PRISMA) guidelines were followed during the preparation of this manuscript15. We specified the inclusion criteria, methods of searching, and analysis in advance. The methods and analyses were conducted in strict accordance to the guidelines of the Cochrane Handbook for Systematic Reviews of Interventions and the Methods Guide for Comparative Effectiveness Reviews16,17.\n\nStudies that fit all of the following criteria were included in the meta-analysis:\n\n(1) Population: Studies whose population was patients with idiopathic Parkinson’s disease (diagnosed using the UK Parkinson’s Disease Society Brain Bank Criteria)18 and including all stages of Parkinson’s disease (mid to late stages)\n\n(2) Intervention: Studies where patients receive safinamide as an experimental drug (all doses are considered) and continue receiving dopamine agonist treatment\n\n(3) Comparator: Studies where the control group received a placebo\n\n(4) Study design: Studies that were described as prospective randomized controlled trials\n\nStudies were excluded based on the following criteria:\n\n(1) Studies using drugs other than safinamide as experimental drugs\n\n(2) Studies not using safinamide ass add-on therapy for motor fluctuations in Parkinson’s disease\n\n(3) Animal studies, in vitro studies, case reports/case series, conference abstracts, or review articles\n\n(4) All studies other than randomized controlled trials (case reports, conference abstracts, and review articles)\n\n(5) Studies unavailable in the English language.\n\nA computer literature search was performed of online databases: PubMed, EMBASE, ClinicalTrial.gov and the Cochrane Library from 1960 to the end of August 2019 (the time of the last search) using the following keywords: (“Safinamide”[All Fields]) AND (“Parkinson disease”[MeSH Terms] OR (“Parkinson”[All Fields] AND “Disease”[All Fields]) OR “Parkinson disease”[All Fields] OR (“Parkinson’s”[All Fields] AND “Disease”[All Fields]) OR “PD”[All Fields]). No restrictions by language or publication period were used.\n\nAfter removal of duplicate articles, two reviewers (M.H and R.G) screened a spreadsheet of titles and abstracts independently using Microsoft Excel 2013 (windows version). Full text studies selected were examined independently by the same two reviewers, the third reviewer (A.N) solve any disagreement by discussion with the main author before the final selection. The independent reviewers are acknowledged for their generous help in searching, screening, and data extraction processes. We did not need to contact any study investigator for further clarification.\n\nAn online data extraction sheet was constructed. One independent reviewer (M.H) extracted the data from included studies and entries were checked by the main author. The data extraction form included the following domains: 1) study ID; 2) year of publication; 3) country; 4) study design (randomized controlled trials only); 5) follow-up duration; 6) safinamide dose; 7) population definition; 8) inclusion and exclusion criteria; 9) sample size; 10) baseline characteristics; 11) available data of outcome measures (pre, post, and change from baseline); and 12) quality assessment domains. A copy of data extraction form is available as extended data19.\n\nCochrane risk of bias assessment tool was used to assess the risk of bias in randomized controlled trials. We assessed the following risks: 1) Selection bias; 2) performance bias; 3) detection bias; 4) attrition bias; 5) reporting bias; and 6) any other source of bias that might have influenced the study data20. One reviewer (A.N) and the main author rated each domain separately as low, high or unclear risk of bias. We used Review Manager software (RevMan 5.3) to summarize the risk of bias of included randomized controlled trials.\n\nThe efficacy of drugs treating motor complications in Parkinson’s disease was assessed for the following outcomes:\n\n(1) Unified Parkinson’s Disease Rating Scale part three (motor part) (UPDRS III): The unified Parkinson’s disease rating scale21 is a reliable score of four parts to assess the severity of Parkinson’s symptoms. Part three indicates the motor score, which is the main measure for motor function in Parkinson’s disease patients.\n\n(2) Patient-reported diaries: Patient diaries gave information about the duration of the following motor outcomes: \"on time with non-troublesome dyskinesia\", which means the duration of absence of dyskinesia associated with the long term usage of Parkinson’s dopaminergic drugs such as levodopa and \"off-time\", which is the duration of returning motor and non-motor symptoms of Parkinson’s disease, even with the use of levodopa and other antiparkinsonian drugs.\n\n(3) Dyskinesia Rating Scale (DRS): Long term usage of dopaminergic drugs leads to involuntary motor movements. This scale is one of the best scales to assess these motor complications22. It measures the following outcomes: \"on time dyskinesia\" and \"off-time dyskinesia\". Additionally, it gives recommendations for descriptions of each type of involuntary movement that can be used when talking with people affected by Parkinson’s.\n\n(4) Clinical Global Impression scale – Severity of Illnes (CGI-S): A seven-point scale used to measure symptom severity, efficacy of the treatments, and treatment response in studies containing patients with mental health issues23.\n\n(5) Unified Parkinson’s Disease Rating Scale part two (UPDRS II): UPDRS is the most commonly used scale to assess the clinical condition of Parkinson’s disease. This is the second part of UPDRS scale, which is used to evaluate the activities of daily life (ADLs) such as hygiene, speech, dressing, and swallowing24.\n\n(6) Parkinson’s Disease Questionnaire (PDQ-39): A self-reported questionnaire with 39 items25. This questionnaire is mainly used to evaluate the difficulties Parkinson’s disease patients face in eight quality of life dimensions, including ADLs, cognition, attention, working memory, depression, social support, social relationship, and functional mobility.\n\n(7) Mini-Mental State Examination (MMSE) scale: A 30-point questionnaire used mainly to evaluate cognitive function. Its usage includes the following: estimating disease progression, severity of impairment of cognitive functions, and documenting the response of mental ill patients to treatment26.\n\n(8) Hamilton Depression Rating Scale (HAM-D): A 21-item test widely used in clinical practice and pharmaceutical trials to assess depressive symptoms27.\n\nSince all the data in the study are continuous data, each efficacy measure is reported as mean difference (MD) between the two groups from the baseline to endpoint, along with its standard error (SE). Both were pooled using the DerSimonian-Laird random effect model. In the case of studies reporting data at multiple time points, the last endpoint was considered.\n\nThe overall MD was interpreted with the consideration that efficacy measures are in different directions; an improvement in \"on time without troublesome dyskinesia\" would be indicated by an increased MD, while an improvement in UPDRS III, “off-time”, UPDRS II, DRS, PDQ-39, MMSE, and HAM-D scores would be indicated by a decreased MD.\n\nThe proportion of risk ratio (RR) was used to pool the adverse events reported in the studies to the total number in each group between the two groups in the DerSimonian-Laird random effect model. To examine heterogeneity of studies, forest plot visual inspection was used and assessed using the Cochrane Q and I2 tests using RevMan version 5.3 for windows.\n\nA few studies, such as as Stoochi (2004), did not report the MD between the safinamide group and placebo group so it was calculated using the following calculation: [MD = MD experimental – MD placebo]. Standard error was calculated from the standard deviation [standard error = standard deviation⁄√n], 95% confidence interval [(upper limit – lower limit) ⁄3.92], or 90% CI [(upper limit − lower limit) ⁄3.29]. For studies and groups with a sample size of less than 60 patients, the numbers (3.92 and 3.29) were substituted by a value from the table of t distributions with degrees of freedom equal to the group sample size minus one.\n\nFunnel plots was used to explore the publication bias across studies and to show the relationship between effect size and precision. The evidence of publication bias was assessed using the following: 1) Egger’s regression test, and 2) the Begg and Mazumdar rank correlation test (Kendall’s tau).\n\nWe performed all the analysis and calculations in this meta-analysis using Review Manager software version 5.3 (RevMan 5.3).\n\n\nResults\n\nThe literature search resulted in 160 studies. After the complete screening process of titles, abstracts, and full texts, 154 studies did not meet the eligibility criteria and six articles with six randomized controlled trials remained with a total of (2556) patients included in the meta-analysis9–14.\n\nA description of the flow of study selection is shown in the PRISMA flow diagram in Figure 1.\n\nThe follow up duration in the studies ranged from 12 weeks in Stoochi et al. (2004) and Stoochi et al. (2011)9,10 to 24 weeks in the study by Schapira et al. (2012), Borgohain et al. (2013), Schapira et al. (2016), and Borgohain et al. (2014)11–14.\n\nThe daily doses of safinamide received in the studies included in the meta-analysis ranged from 40mg in the study by Stoochi et al. (2004)9 to 200mg in studies by Stoochi et al. (2011)10 and Schapira et al. (2012)11. The population of all studies was homogenous and remained on the dopaminergic treatment during the entire study period.\n\nAll patients enrolled in the studies were diagnosed with Parkinson’s disease according to UK Parkinson’s Disease Society Brain Bank Criteria. The criteria of patients excluded from studies were: 1) history of psychiatric disorders, 2) severe and progressive medical illness, 3) patients with dementia, 4) severe dyskinesia. Summary and baseline characteristics of populations of these studies are shown in Table 1.\n\n*Continuous outcomes presented as mean (SD). RCT, randomized controlled trial; UPDRS III; Unified Parkinson’s Disease Rating Scale part three; CGI-S, Clinical Global Impression scale – Severity of Illness; MMSE, Mini-Mental State Examination; HAM-D, Hamilton Depression Rating Scale; NR, not reported.\n\nThe Cochrane risk of bias assessment tool was used to assess the quality of included studies. All included studies had a low risk of bias in terms of random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting except Schapira et al. (2012)11, which had a high risk of bias for random sequence generation as the randomization method was not reported and for the blinding of outcome assessment as the method of blinding was not reported, and Stoochi et al. (2004)9, which had a high risk for incomplete outcome data because there was no intention to treat analysis for missed or withdrawn patients mentioned in the study. The summary of risk of bias domains is shown in Figure 2.\n\nOff-time. The overall MD between the two groups from baseline to endpoint in terms of change in \"off-time\" favored safinamide over placebo (MD -0.72 h, 95% CI [-0.89 to -0.56], Figure 3A). Pooled studies were homogenous (P=0.42).\n\nForest plot of the mean difference and 95% confidence interval of the following outcome; (A) Off time, (B) On-time without troublesome dyskinesia, (C) UPDRS-III, (D) UPDRS-II.\n\nOn time without troublesome dyskinesia. The overall MD between the two groups from baseline to endpoint in terms of change in \"on time without troublesome dyskinesia\" favored safinamide over placebo (MD 0.71 h, 95% CI [0.52 to 0.90], Figure 3B). Pooled studies were homogenous (P=0.54).\n\nUPDRS III. The overall MD between the two groups from baseline to endpoint in terms of change in \"UPDRS III\" favored safinamide over placebo (MD -1.83, 95% CI [-2.43 to -1.23], Figure 3C). Pooled studies were homogenous (P=0.80).\n\nUPDRS II. The overall MD between the two groups from baseline to endpoint in terms of change in \"UPDRS II\" favored safinamide over placebo (MD -0.69, 95% CI [-1.03 to -0.36], Figure 3D). Pooled studies were homogenous (P=0.26).\n\nDRS score. The overall MD between the two groups from baseline to endpoint in terms of change in \"DRS score\" did not favor either of the two groups (MD -0.14 h, 95% CI [-0.36 to 0.08], Figure 4E). Pooled studies were homogenous (P=0.15).\n\nForest plot of the mean difference and 95% confidence interval of the following outcomes; (E) DRS, (F) CGI-S, (G) PDQ-39, (H) HAM-D, (L) MMSE.\n\nCGI severity. The overall MD between the two groups from baseline to endpoint in terms of change in \"CGI severity\" favored safinamide over placebo (MD -0.18 h, 95% CI [-0.24 to -0.12], Figure 4F). Pooled studies were homogenous (P=0.70).\n\nPDQ-39. The overall MD between the two groups from baseline to endpoint in terms of change in \"PDQ-39\" favored safinamide over placebo (MD -1.59 h, 95% CI [-2.56 to -0.61], Figure 4G). Pooled studies were homogenous (P=0.35).\n\nHAM-D. The overall MD between the two groups from baseline to endpoint in terms of change in \"HAM-D\" favored safinamide over placebo (MD -0.35 h, 95% CI [-0.64 to -0.06], Figure 4H). Pooled studies were homogenous (P=0.88).\n\nMMSE. The overall mean difference between the two groups from baseline to endpoint in terms of change in \"MMSE\" favored safinamide over placebo (MD -0.16 h, 95% CI [-0.36 to -0.05], Figure 4L). Pooled studies were homogenous (P=0.79).\n\nAdverse events. The following adverse events were reported in the included studies: Back pain, cataeacts, dizziness, hypertension, dyskinesia, headaches, and worsening of Parkinson’s disease, as well as discontinuation due to treatment emergent adverse events (TEAEs), including serious TEAEs, serious drug-related TEAEs, and any TEAEs.\n\n(A) Back pain\n\nSeven studies reported back pain. The pooled meta-analysis did not favor either of the two groups (RR 0.75, 95% CI [0.56 to 1.02], Figure 5A). Pooled studies were homogenous (P=0.46).\n\nForest plot presentation of Meta-Analysis for the Following Adverse Events of Safinamide; (A) Back pain, (B) Cataract, (C) Dizziness, and (D) Hypertension.\n\n(B) Cataracts\n\nSix studies reported cataracts. The pooled meta-analysis did not favor either of the two groups (RR 0.95, 95% CI [0.69 to 1.31], Figure 5B). Pooled studies were homogenous (P=0.81).\n\n(C) Dizziness\n\nFive studies reported dizziness. The pooled meta-analysis did not favor either of the two groups (RR 0.69, 95% CI [0.36 to 1.32], Figure 5C). Pooled studies were homogenous (P=0.85).\n\n(D) Hypertension\n\nSeven studies reported hypertension. The pooled meta-analysis did not favor either of the two groups (RR 1.42, 95% CI [0.99 to 2.03], Figure 5D). Pooled studies were homogenous (P=0.82).\n\n(E) Dyskinesia\n\nSeven studies reported dyskinesia. The pooled meta-analysis showed increase of dyskinesia in patients receiving placebo compared to safinamide (RR 1.50, 95% CI [1.25 to 1.80], Figure 6E). Pooled studies were homogenous (P=0.10).\n\nForest plot presentation of Meta-Analysis for the Following Adverse Events of Safinamide; (E) Dyskinesia, (F) Headache, (G) Worsening Parkinson’s disease, and (H) TEAE’s leads to discontinuation.\n\n(F) Headache\n\nFive studies reported headaches. The pooled meta-analysis did not favor either of the two groups (RR 1.10, 95% CI [0.79 to 1.53], Figure 6F). Pooled studies were homogenous (P=0.70).\n\n(G) Worsening Parkinson’s disease\n\nSeven studies reported patients with worsening of Parkinson’s disease during the study. The pooled meta-analysis did not favor either of the two groups (RR 0.82, 95% CI [0.65 to 1.03], Figure 6G). Pooled studies were homogenous (P=0.54).\n\n(H) TEAEs leading to discontinuation\n\nNine studies reported the number of patients with TEAEs leading to discontinuation of the study. The pooled meta-analysis did not favor either of the two groups (RR 1.05, 95% CI [0.75 to 1.46], Figure 6H). Pooled studies were homogenous (P=1.00).\n\n(I) Serious drug-related TEAEs\n\nThree studies reported the number of patients with serious drug-related adverse events. The pooled meta-analysis did not favor either of the two groups (RR 0.72, 95% CI [0.32 to 1.62], Figure 7I). Pooled studies were homogenous (P=0.32).\n\nForest plot presentation of Meta-Analysis for the Following Adverse Events of Safinamide; (I) serious drug related TEAE’s, (L) any study drug related TEAE’s, (M) any TEAE’s, and (N) Serious TEAE’s.\n\n(L) Any drug-related TEAEs\n\nFive studies reported the number of patients with any drug-related TEAEs. The pooled meta-analysis showed an increase in the number of patients with any drug-related adverse events in the placebo group compared to the safinamide group (RR 1.19, 95% CI [1.03 to 1.36], Figure 7L). Pooled studies were homogenous (P=0.36).\n\n(M) Any TEAEs\n\nSeven studies reported the number of the patients with any TEAEs. The pooled meta-analysis did not favor either of the two groups (RR 0.98, 95% CI [0.93 to 1.02], Figure 7M). Pooled studies were homogenous (P=0.88).\n\n(N) Serious TEAEs\n\nNine studies reported the number of the patients with serious TEAEs. The pooled meta-analysis did not favor either of the two groups (RR 1.02, 95% CI [0.81 to 1.28], Figure 7N). Pooled studies were homogenous (P=0.38).\n\nAs showed in Figure 8, funnel plots of UPDRS III, off-time, on-time without troublesome dyskinesia, and UPDRS II show no significant publication bias across studies.\n\nFunnel plots for publication bias for (A) UPDRS III, (B) Off-time, (C) on-time without troublesome dyskinesia, and (D) UPDRS II.\n\n\nDiscussion\n\nThe pooled meta-analysis of six studies provides a class I evidence that using safinamide as add-on therapy for Parkinson’s disease is very effective and well tolerated. The meta-analysis shows that safinamide improves motor fluctuations, which is a main side effect of anti-Parkinson’s medications, as reported by patient diaries and measured by \"on time without troublesome dyskinesia\", \"off-time\", and UPDRS III score. This novel drug is also improving the quality of life of Parkinson’s disease patients, as measured by the UPDRS II scale, the PDQ-39 questionnaire, HAM-D, and MMSE.\n\nRegarding tolerability, safinamide is a well-tolerated drug and despite increasing the risk of some adverse events such as dyskinesia, which was higher in the safinamide group than the placebo10–14, the pooled meta-analysis of RRs of adverse events did not show any statistical significance between the two groups of comparison.\n\nThe results obtained from the meta-analysis are consistent with the results of the previous randomized controlled trials in terms of outcomes measuring motor fluctuations and quality of life. “On time without troublesome dyskinesia” and “off-time” are the main outcomes to evaluate motor fluctuations and were mentioned in Schapira et al. (2016) with (100 mg daily dose), Borgohain et al. (2013 and 2014) with doses of 50 and 100 mg daily. In all the previously mentioned studies, “on time without troublesome dyskinesia” favored the safinamide group over the placebo group12–14. In addition, the mean difference of “off-time” in all studies favored the safinamide group and this result was consistent with the pooled meta-analysis of included studies12–14.\n\nThe UPDRS III is a very important scale for evaluating the motor symptoms in Parkinson’s patients. Despite the results of Stoochi et al. (2011) (200mg daily dose), Stoochi et al. (2004) (40mg and 70mg daily doses), and Schapira et al. (2012) (pooled doses of 100mg and 200mg daily doses)9–11, which showed no statistical significance between safinamide and placebo groups, the pooled meta-analysis showed that the UPDRS III score favors safinamide over placebo. The results of the DRS score in the included studies did not favor safinamide over placebo and the pooled meta-analysis did not favor one group either.\n\nQuality of life in Parkinson’s disease patients is measured by UPDRS II, PDQ-39, HAM-D, and MMSE scores. The studies that mentioned the outcomes of quality of life showed results consistent with the pooled meta-analysis that the use of safinamide is preferable to placebo10,11,14, except MMSE scores which were mentioned in Schapira et al. (2016), and Schapira et al. (2012)11,14 and showed no statistical significance between safinamide and placebo in both the included studies and the pooled meta-analysis.\n\nThe strengths of the meta-analysis are the following: 1) multiple search engines were searched and all the possible sources of studies to be included were covered; 2) clear eligibility criteria were provided; 3) multiple reviewers revised every step to ensure accuracy; 4) during the preparation of this manuscript, the PRISMA guidelines were followed; 5) the study was conducted according to the guidelines of the Cochrane Handbook for Systematic Reviews of Interventions in a strict way; 6) the randomized controlled trials included data of high validity and acceptable quality, as indicated by the risk of bias assessment.\n\nThe meta-analysis limitations are the following: A) some studies, such as as Stoochi et al. (2004), Stoochi et al. (2011), and Schapira et al. (2012)9–11 did not mention outcomes such as “on-time without troublesome dyskinesia” and “off-time”, which are important measurements for motor symptoms evaluation; B) there was no standardization in the reporting of adverse events in the included studies; C) there was a high risk of bias in some studies, namely as Stoochi et al. (2004)9 and Schapira et al. (2012)11.\n\nBased on the results of the study, future randomized controlled trials with different doses are recommended to investigate the efficacy of safinamide for Parkinson’s disease patients with motor fluctuations as a side effect of anti-Parkinson’s medications.\n\n\nConclusions\n\nDespite the evidence provided by this meta-analysis, demonstrating the efficacy of safinamide as add-on therapy for treatment of motor complications of anti-Parkinson’s disease medications, future studies are still needed to confirm the safety and efficacy of this novel drug.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nOpen Science Framework: A systematic review and meta-analysis of safety and efficacy of Safinamide for motor fluctuations in patients with Parkinson’s disease. https://doi.org/10.17605/OSF.IO/T6H9J19\n\nThis project contains the following extended data:\n\n- Data extraction form.xlsx\n\n- Spreadsheets in .xlsx format containing extracted data for drug efficacy outcomes including adverse events\n\nOpen Science Framework: A systematic review and meta-analysis of safety and efficacy of Safinamide for motor fluctuations in patients with Parkinson’s disease. https://doi.org/10.17605/OSF.IO/T6H9J19\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe author would like to thank Dr. Ahmed Negida (first reviewer), Randa Gamal (second reviewer), and Mohamed Helmy (third reviewer) for their generous support and guidance through the procedure of completing this manuscript (searching, screening, and data extraction processes).\n\n\nReferences\n\nMarras C, Beck JC, Bower JH, et al.: Prevalence of Parkinson’s disease across North America. NPJ Parkinsons Dis. 2018; 4(1): 21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchapira AH, Emre M, Jenner P, et al.: Levodopa in the treatment of Parkinson’s disease. Eur J Neurol. 2009; 16(9): 982–9. PubMed Abstract \| Publisher Full Text\n\nFerreira JJ, Katzenschlager R, Bloem BR, et al.: Summary of the recommendations of the EFNS/MDS-ES review on therapeutic management of Parkinson’s disease. Eur J Neurol. 2013; 20(1): 5–15. PubMed Abstract \| Publisher Full Text\n\nPoewe WH, Lees AJ, Stern GM: Low-dose L-dopa therapy in Parkinson’s disease: a 6-year follow-up study. Neurology. 1986; 36(11): 1528–30. PubMed Abstract \| Publisher Full Text\n\nRinne UK: Treatment of Parkinson’s disease: problems with a progressing disease. J Neural Transm. 1981; 51(1-2): 161–74. PubMed Abstract \| Publisher Full Text\n\nObeso JA, Olanow CW, Nutt JG: \"Levodopa motor complications in Parkinson’s disease.\" Trends Neurosci. 2000; 23(10 Suppl): S2–S7. PubMed Abstract \| Publisher Full Text\n\nMarzo A, Dal Bo L, Monti NC, et al.: Pharmacokinetics and pharmacodynamics of safinamide, a neuroprotectant with antiparkinsonian and anticonvulsant activity. Pharmacol Res. 2004; 50(1): 77–85. PubMed Abstract \| Publisher Full Text\n\nSchapira AH: Safinamide in the treatment of Parkinson’s disease. Expert Opin Pharmacother. 2010; 11(13): 2261–8. PubMed Abstract \| Publisher Full Text\n\nStocchi F, Arnold G, Onofrj M, et al.: Improvement of motor function in early Parkinson disease by safinamide. Neurology. 2004; 63(4): 746–8. PubMed Abstract \| Publisher Full Text\n\nStocchi F, Borgohain R, Onofrj M, et al.: A randomized, double-blind, placebo-controlled trial of safinamide as add-on therapy in early Parkinson’s disease patients. Mov Disord. 2012; 27(1): 106–12. PubMed Abstract \| Publisher Full Text\n\nSchapira AH, Stocchi F, Borgohain R, et al.: Long-term efficacy and safety of safinamide as add-on therapy in early Parkinson’s disease. Eur J Neurol. 2013; 20(2): 271–80. PubMed Abstract \| Publisher Full Text\n\nBorgohain R, Szasz J, Stanzione P, et al.: Randomized trial of safinamide add-on to levodopa in Parkinson’s disease with motor fluctuations. Mov Disord. 2014; 29(2): 229–37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBorgohain R, Szasz J, Stanzione P, et al.: Two-year, randomized, controlled study of safinamide as add-on to levodopa in mid to late Parkinson’s disease. Mov Disord. 2014; 29(10): 1273–80. PubMed Abstract \| Publisher Full Text\n\nSchapira AH, Fox SH, Hauser RA, et al.: Assessment of Safety and Efficacy of Safinamide as a Levodopa Adjunct in Patients With Parkinson Disease and Motor Fluctuations: A Randomized Clinical Trial. JAMA Neurol. 2017; 74(2): 216–224. PubMed Abstract \| Publisher Full Text\n\nLiberati A, Altman DG, Tetzlaff J, et al.: The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration. PLoS Med. 2009; 6(7): e1000100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHiggins JP, Green S: Cochrane handbook for systematic reviews of interventions. John Wiley & Sons. 2011. Publisher Full Text\n\nWhitlock EP, Lopez SA: Methods Guide for Comparative Effectiveness Reviews.\n\nClarke CE, Patel S, Ives N, et al.: Clinical effectiveness and cost-effectiveness of physiotherapy and occupational therapy versus no therapy in mild to moderate Parkinson’s disease: a large pragmatic randomized controlled trial (PD REHAB). Southampton (UK): NIHR Journals Library. (Health Technology Assessment, No. 20.63.) Appendix 1, UK Parkinson’s Disease Society Brain Bank Diagnostic Criteria. 2016. Reference Source\n\nAziz MA: A systematic review and meta-analysis of safety and efficacy of safinamide for motor fluctuations in patients with Parkinson’s disease. 2019. http://www.doi.org/10.17605/OSF.IO/T6H9J\n\nHiggins JP, Altman DG, Gøtzsche PC, et al.: The Cochrane Collaboration’s tool for assessing risk of bias in randomised trials. BMJ. 2011; 343: d5928. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMovement Disorder Society Task Force on Rating Scales for Parkinson’s Disease. The unified Parkinson’s disease rating scale (UPDRS): status and recommendations. Mov Disord. 2003; 18(7): 738–50. PubMed Abstract \| Publisher Full Text\n\nGoetz CG, Nutt JG, Stebbins GT: The unified dyskinesia rating scale: presentation and clinimetric profile. Mov Disord. 2008; 23(16): 2398–403. PubMed Abstract \| Publisher Full Text\n\nGuy W: ECDEU assessment manual for psychopharmacology. US Department of Health, and Welfare. 1976: 534–537. Reference Source\n\nRamaker C, Marinus J, Stiggelbout AM, et al.: Systematic evaluation of rating scales for impairment and disability in Parkinson’s disease. Mov Disorder. 2002; 17(5): 867–76. PubMed Abstract \| Publisher Full Text\n\nJenkinson C, Fitzpatrick R, Peto V, et al.: The Parkinson’s Disease Questionnaire (PDQ-39): development and validation of a Parkinson’s disease summary index score. Age Ageing. 1997; 26(5): 353–7. PubMed Abstract \| Publisher Full Text\n\nPangman VC, Sloan J, Guse L: An examination of psychometric properties of the mini-mental state examination and the standardized mini-mental state examination: implications for clinical practice. Appl Nurs Res. 2000; 13(4): 209–13. PubMed Abstract \| Publisher Full Text\n\nHamilton M: A rating scale for depression. J Neurol Neurosurg Psychiatry. 1960; 23: 56–62. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "61382", "date": "24 Mar 2020", "name": "Fabrizio Stocchi", "expertise": [ "Reviewer Expertise Movement disorders", "Parkinson’s disease", "neuropharmacology", "clinical trials." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors published a systematic review and meta-analysis of safety and efficacy of safinamide for motor fluctuations in patients with Parkinson's disease. There are other review on safinamide but the meta-analysis can be interested. However the paper must be revised addressing some important issues:\nIntroduction:\nNon-motor symptoms should be better addressed and ref. implemented and revised. Within the other treatment, COMTI are not mentioned\n\nThe meta-analysis is well conducted and gave a number of good information, but there are some problems:\n\nThe studies were grouped according to the presence of the end-point. However for UPDRS III studies with different population were grouped together, moreover in some of these studies UPDR III was the primary end-point but for the other studies it was a secondary end-point. UPDRS scored in an early population give information about the effect of the drug on symptoms, in fluctuators it does not.\n\nOther parameter were influenced by the dose in the original study (PDQ39, UPDRS II) but putting all the patents together this effect does not emerge giving the false impression that the drug is always efficacious. These aspects should be carefully addressed and discussed.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] }, { "id": "61383", "date": "27 Apr 2020", "name": "Rupam Borgohain", "expertise": [ "Reviewer Expertise Movement disorders", "Botulinum toxin", "Deep brain stimulation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors published a systematic review and meta-analysis of safety and efficacy of safinamide for motor fluctuations in patients with Parkinson's disease. The meta-analysis is the first one and has been conducted well. It demonstrates the efficacy and safety of safinamide in Parkinson's disease and its value in patients with dyskinesias. Safinamide can be used in early PD as a single drug or in combination with dopamine agonist. It can be an add on in mid-late PD for motor fluctuations. The methodology states that the studies which did not use safinamide as an add on therapy for motor fluctuations in PD would exclude some studies included. However, the studies included in the meta-analysis include patients with early PD as well as those with mid-late PD. Early PD patients do not have motor fluctuations, dyskinesias, are less likely to have depression, cognitive impairment and thus this subset may undervalue the actual effect of safinamide.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] }, { "id": "58806", "date": "06 May 2020", "name": "Ahmed Negida", "expertise": [ "Reviewer Expertise I'm a medical doctor", "holding an MD degree", "and currently doing a Ph.D. in Parkinson's Disease treatment (the same scope of this manuscript). I have several similar publications in the field." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes a systematic review and meta-analysis of published studies on the Safenamide for the treatment of motor fluctuations in Parkinson's Disease.\n\nFirst, the topic is interesting because currently, there is an unmet clinical need to find potential agents that decrease the motor fluctuations and the LED in PD midstage and late-stage PD.\n\nSecond, the methodology is clear and in accordance with the Cochrane handbook guidelines, while the manuscript is well-written in compliance with the standard reporting guidelines (PRISMA statement) - endorsed by the ICMJEs.\nThe statistical analysis was properly done and the data analysis interpretation and discussion are sufficient.\nI do not find any major concerns in the article; I believe it worth the publication. No further changes are required from my point of view.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-2078
https://f1000research.com/articles/8-2077/v1	10 Dec 19	{ "type": "Research Article", "title": "Expression of micro-RNAs miR-31, miR-146a, miR-181c and miR-155 and their target gene IL-2 are altered in schizophrenia: a case-control study", "authors": [ "Hovsep Ghazaryan", "Roksana Zakharyan", "Martin Petrek", "Zdenka Navratilova", "Andranik Chavushyan", "Eva Novosadova", "Arsen Arakelyan", "Hovsep Ghazaryan", "Roksana Zakharyan", "Martin Petrek", "Zdenka Navratilova", "Andranik Chavushyan", "Eva Novosadova" ], "abstract": "Background: Schizophrenia is a severe psychiatric disorder with a heterogeneous clinical phenotype. The association of interleukins and other cytokines and their receptors with schizophrenia has been previously reported. Additionally, a number of studies have reported altered mico-RNA (miRNA) expression in schizophrenia and other psychiatric disorders. The aim of our study was to explore the possible association of miR-31, miR-146a, miR-181c and miR-155 with schizophrenia pathogenesis, as well as their link to IL2 gene expression in disease. Methods: For this case-control study, 225 patients with paranoid schizophrenia and 225 sex- and age-matched controls with no family history of schizophrenia were recruited. The expression of studied miRNAs and the IL2 gene was measured using qPCR. DNA samples of all patients and controls were genotyped for IL2 rs2069778 single nucleotide polymorphism (SNP) using PCR with sequence specific primers (PCR-SSP). Statistical analyses include the Mann-Whitney U-test and Fischer’s exact test. Results: All studied miRNAs were over-expressed in schizophrenic patients IL2 gene expression was down-regulated in schizophrenic patients. The IL2 rs2069778 SNP is not associated with schizophrenia but regulates expression of the IL2 gene. Conclusions: Over-expression of studied miRNAs and down-regulation of IL2 gene expression may be considered as genetic risk factors for chronic schizophrenia. Abnormalities in studied miRNA expressions result in the deregulation of the T-cell receptor signaling pathway in schizophrenia.", "keywords": [ "schizophrenia", "miRNA", "interleukin-2", "cytokines", "SNP", "expression", "pathway" ], "content": "Introduction\n\nSchizophrenia (OMIM code: 181500) is a severe psychiatric disorder with a heterogeneous clinical phenotype1. While the etiology of this disorder remains largely unknown, it has become evident that immune-inflammatory processes play an important multilevel role in disease development and progression2–4. Both cellular and humoral components of the innate and adaptive immune system were shown to be altered at different stages of disease development, both locally within the central nervous system as well as at a systemic level. Cytokines and chemokines are essential signal mediators of the immune system that modulate and guide immune/inflammatory responses as well as perform a wide range of other functions related to cell survival, proliferation, differentiation and migration5. Considering their biological importance, cytokines, especially interleukins (IL), have received considerable attention in the context of schizophrenia. The association of interleukins and other cytokines and their receptors with schizophrenia has been previously reported both on the level of genetic variants as well as the levels of gene expression and protein abundance, indicating their essential role in disease predisposition, development, progression and treatment response6–9.\n\nIL-2 controls a wide range of biological activities, largely depending on the biological context. It is essential for T lymphocyte proliferation and differentiation, but it is also implicated in the generation and maintenance of regulatory T (Treg) cells10,11. The role of the IL2 gene in schizophrenia is still unclear and conflicting. However, previous studies, including our own findings, suggest the involvement of IL-2 in the pathogenesis of schizophrenia. Decreased lymphocyte production of IL-2 and increased IL-2 receptors have been reported previously12–16. Interleukin-2 receptor gamma (IL2RG) is an important signaling component of receptors for many cytokines, including IL-2, -4, -7, -9, -15 and -2113. Moreover, the IL2RG gene is over-expressed in the blood of schizophrenia patients13. On the other hand, other groups reported increased IL-2 serum levels in schizophrenia17–19. Some IL2 genetic polymorphisms were also reported to be associated with schizophrenia20,21. Expression of the IL2 gene is controlled at multiple layers. The IL2 gene contains at least two cis elements for transcript stability regulation, located in both the 3’ and 5’ untranslated regions (UTRs)22,23. Single nucleotide polymorphisms (SNPs) in the promoter region of IL2 influence the expression levels of this cytokine. Finally, expression of IL2 is also regulated by micro-RNAs24.\n\nMicro-RNAs (miRNAs) are class of small, non-coding RNAs (comprised of about 22 nucleotides). MiRNAs are found in animals, plants and some viruses. They function in the regulation of gene expression at posttranscriptional level and RNA silencing25–27. As miRNAs are involved in the normal functioning of eukaryotic cells, deregulation of miRNAs has become associated with disease. There is a manually curated “miR2Disease” database, which aims to provide a comprehensive resource of microRNA deregulation in various human diseases28. Also, a number of studies have reported altered miRNA expression in schizophrenia, bipolar disorder and major depression and anxiety disorders29–32.\n\nThe aim of our study was to explore the possible association of miR-31, miR-146a, miR-181c and miR-155 with schizophrenia pathogenesis, as well as their link to IL2 gene expression in disease. This study is the first to report genetic association between schizophrenia and mentioned above miRNAs; however, several studies have reported a role for the IL2 gene in schizophrenia. All of these four micro-RNAs play a major role in regulating expression of the cytokine network. Particularly, miR-31, miR-146a and miR-181c are regulators of IL2 gene expression33–35, while miR-155 expression is greatly enhanced following stimulation of macrophages and dendritic cells by Toll-like receptors36. We also studied the possible association of the IL2 rs2069778 SNP genotype with IL2 and miRNA levels. This SNP was chosen due to its high minor allele frequency, clinical significance in autoimmune diseases37, as well as its location near the regulatory elements of the IL2 gene.\n\n\nMethods\n\nInformed written consent was obtained from all study participants. The study has been approved by the Ethical Committee of the Institute of Molecular Biology of the National Academy of Sciences RA (IRB00004079, IORG0003427).\n\nThis case-control study was conducted from January 2016 to February 2017. A total of 225 patients with paranoid schizophrenia (SCZ) and 225 sex- and age-matched controls (CTRL) with no family history of schizophrenia were involved in this study (Table 1). This was the maximum available number of schizophrenia patients in Armenia who agreed to participate in this study. From these subjects, 61 patients and 60 controls were tested for micro-RNA expression and 66 patients and 99 controls for IL2 gene expression. There was no specific criteria for dividing subsets in this study; subjects were divided into groups according to availability of biological material (DNA and RNA). All subjects were genotyped for this study.\n\nParanoid schizophrenia (OMIM code: 181500, ICD-10-CM code: F20.0, DSM-5 code: 295.90) was diagnosed by two independent psychiatrists. Schizophrenia patients were recruited from the clinics of the Psychiatric Medical Center of the Ministry of Health of the Republic of Armenia (MH RA). Healthy subjects with any psychiatric illness during their lifetime, any serious endocrine or neurological disorder, any treatment or medical condition known to affect the brain or meeting the DSM-5 criteria for intellectual disability38 were excluded from this study. Exclusion criteria for all study subjects included any treatment with immune-modulating drugs and serious medical disorder.\n\nHealthy control subjects were recruited among the blood donors of the Erebouni Medical Center (MH RA) and were interviewed by psychiatrists.\n\nA total of 10 ml of peripheral blood was collected in EDTA containing tubes (5ml for RNA and 5ml for DNA isolation) from each study participant.\n\nPeripheral blood mononuclear cells were isolated from whole blood using the following protocol, as described in 13: 10 ml of Red Cell Lysis Buffer (RCLB) (containing 0.144M ammonium chloride, 1 mM sodium bicarbonate) was added to 5 ml of fresh blood. After 5 minutes, the mixture was centrifuged at 1000g for 10 minutes, the supernatant was discarded and the pellet was gently rinsed with RCLB. The pellet was re-suspended in 5 ml of the RCLB buffer and centrifuged at 1000g for 10 minutes. The final purified pellet was stored in RNAlater (Ambion, Austin, TX, USA) at -20°C for later use. Total RNA was extracted using High Pure miRNA Isolation Kit (Cat. No. 05080576001 Roche Applied Science, Penzberg, Germany) according to the manufacturer’s instructions. The quantity and quality of RNA and DNA samples were assessed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol and stored at -80C until further use. The average RNA yield was 25 mg per 5 ml of blood sample.\n\nThe method for genomic DNA isolation was a modification of Miller’s salting-out procedure39 where a chloroform extraction phase is added (2ml of chloroform added to each tube supernatant and centrifuged at 3000g for 10 minutes)40.\n\ncDNA synthesis was performed by reverse transcription (0.5 μg total RNA, total volume of cDNA 20 μl) using Transcriptor First Strand cDNA Synthesis Kit (Cat. No. 04897030001, Roche Applied Science) with anchored dT primers (0.4 μg; ABgene, Waltham, MA, USA) at 47°C for 45 min. cDNA samples were stored at -20°C until further use.\n\nmRNA levels of IL2 (target) and PSMB2 (housekeeping) genes and were measured by quantitative real-time polymerase chain reaction with the Rotor Gene 3000 instrument (Corbett Research, USA). cDNA (5 μl, corresponding to 20 ng calculated on input total RNA) was added to 20 μl PCR-mix. The final reaction mix contained 900 nM of each sense and antisense primer (Roche Applied Science), 100 nM LNA probe (Roche Applied Science), 3.5 mM MgCl2, 200 μM of each dNTP (ABgene), 0,2μl Thermo-Start TAQ polymerase with concentration 5 U/μl and 1x ThermoStart Buffer (ABgene).\n\nqPCR was performed using following thermal cycling conditions: initial denaturation for 15 minutes at 95°C; 40 cycles of denaturation for 20 seconds at 95°C, annealing for 30 seconds at 60°C and extension for 60 seconds at 72°C; and a final extension for five minutes at 72°C.\n\nThe primers and fluorescently-labeled Locked Nucleic Acid (LNA) probe from the Universal Probe Library for the IL2 gene were selected using the Probe Finder web-based software as follows:\n\nIL2 gene:\n\nLeft primer: 5'-AAG TTT TAC ATG CCC AAG AAG G-3'\n\nRight primer: 5'AAG TGA AAG TTT TTG CTT TGA GCT A-3'\n\nProbe: #65\n\nPSMB2 gene:\n\nLeft primer 5'-GTG AGA GGG CAG TGG AAC TC-3'\n\nRight primer 5'-GAA GGT TGG CAG ATT CAG GA-3'\n\nProbe #50\n\nReverse transcription for selected micro-RNAs and measurement of micro-RNA expression by quantitative real-time polymerase chain reaction (RT-PCR) were performed using TaqMan Micro-RNA Assays (see Table 2) and TaqMan Universal PCR Master MIX II (no UNG) (Cat. No. 4440040, Thermo Fisher Scientific, Waltham, USA), according to the manufacturer’s instructions. qPCR was performed using Realist-DX IAB real-time PCR system (GeneTiCA, Czech Republic).\n\nqPCR was performed using following thermal cycling conditions: polymerase activation for 10 minutes at 95°C; 40 cycles of denaturation for 15 seconds at 95°C and annealing/extension for 60 seconds at 60°C.\n\nWe used miR-16 as a housekeeping micro-RNA. Data were expressed as arbitrary units (miR-X/miR-16 ratio). miR-16 is currently one of generally accepted housekeeping micro-RNAs for normalizing micro-RNA expression in blood cells by qRT PCR41. In addition, there are no reports on miR-16 alterations in any psychiatric disease including schizophrenia.\n\nDNA samples of all patients and controls were genotyped for IL2 rs2069778 SNP using a polymerase chain reaction with sequence-specific primers (PCR-SSP)42. The sequences of specific primers were designed based on relevant DNA sequences available in the NCBI GenBank database (RefSeq Accession: NG_016779.1). Primer sequences for the selected SNP were as follows:\n\nReverse standard: 5’-CAC CAC TAC AAA TTC TAC AAA TTC G-3'\n\nReverse mutant: 5’-CAC CAC TAC AAA TTC TAC AAA TTC A-3'\n\nForward constant: 5’-CTG GTG CCA GAA AGA GCT TG-3'\n\nThe presence/absence of allele-specific amplicons were visualized by electrophoresis using 2% agarose gel in 0.5x Tris-Borate-EDTA (TBE) buffer stained with ethidium bromide fluorescent dye. To check the reproducibility of results, randomly selected DNA samples of study subjects (10% of total) were genotyped twice.\n\nGenotyping was carried out at Laboratory of Human Genomics and Immunomics, Institute of Molecular Biology NAS RA (Yerevan, Armenia).\n\nThe Shapiro–Wilk test for normality revealed non-parametric distribution of the obtained data. Therefore, the significance of difference in gene expression levels between each study group was analyzed by the Mann–Whitney U test. p-values less than 0.05 were considered as significant. Statistical analysis was performed using GraphPad Prism (version 5) software. Allele and genotype frequencies were checked for Hardy-Weinberg equilibrium and were in equilibrium. No investigation of potential sources of bias was undertaken.\n\nFor characterization of enriched functions and biological pathways of the studied miRNAs targets, we used miRsystem43, which performs enrichment analyses, accounting both for target genes as well as the levels of individual miRNA expression.\n\n\nResults\n\nIn total, 66 SCZ patients (male/female: 33/33, mean age±S.D.: 51±11.2 years) and 99 healthy controls (male/female: 45/44, mean age±S.D.: 50±13.9 years) participated in IL2 gene expression step44.\n\nWe studied mRNA expression levels of the IL2 gene in schizophrenia and its possible association with the IL2 rs2069778 C/T SNP. The median mRNA expression levels of IL2 in the patient group were significantly lower than in healthy control subjects (patients vs. controls, median [interquartile range]: 0.06889 [0.6499–0.007519] vs. 1.469 [3.858–0.000], p=0.0095) (Figure 1).\n\nFurthermore, analysis revealed a significant difference in IL2 expression between those with and without the IL2 rs2069778 C/T SNP in both the control and schizophrenia groups. Particularly, in schizophrenic patients, IL2 mRNA expression levels were significantly higher in rs2069778*T minor allele carriers (CT+TT) than in CC homozygotes (CC vs. CT+TT, median [interquartile range]: 0.033711 [0.5433–000453] vs. 0.2178 [2.618–0.03726], p=0.0003) (Figure 2). The same difference was found in control groups (CC vs. CT+TT, median [interquartile range]: 1.083 [2.840–0.000] vs. 2.625 [4.966–0.000], p=0.0495) (Figure 2). It is worth noting that expression of IL2 in schizophrenic patients carrying the CC genotype was lower than in corresponding controls (p=0.0216), while the difference in expression between carriers of the T minor allele was not significant (p=0.22).\n\nA total of 61 SCZ patients (male/female: 37/24, mean age±S.D.: 45.4±13.9 years) and 60 healthy controls (male/female: 37/23, mean age±S.D.: 44.5±13.6 years) were tested for micro-RNA expression.\n\nMedian expression levels of all studied miRNAs were significantly higher in schizophrenic patients as compared to healthy controls (Table 3 and Figure 3)45.\n\nFurther analysis indicated that in T allele carriers, miR-181c had significantly lower expression compared to CC homozygous variants, both in schizophrenia patients (TT+CT vs. CC, median [interquartile range]: 1.99 [2.92–1.39] vs. 3.46 [4.84–2.14], p=0.0045) and controls (TT+CT vs. CC, median [interquartile range]: 0.41 [2.41–0.10] vs. 1.90 [5.31–0.59], p=0.011) (Figure 4).\n\nCC – homozygous for rs2069778 SNP, TT+CT – carrier for rs2069778 SNP.\n\nInterestingly, in T allele carriers, miR-31 also had significantly lower expression compared to CC homozygous variants in controls (TT+CT vs. CC, median [interquartile range]: 0.09 [1.61–0.03] vs. 1.84 [3.35–0.48], p=0.0015) but in schizophrenia patients there was no significant difference (TT+CT vs. CC, median [interquartile range]: 3.93 [7.74–2.91] vs. 5.03 [8.12–3.16], p=0.36) (Figure 4). We have not found any association between IL2 rs2069778 variants and expressions of miR-155 and miR-146a.\n\nOur analysis demonstrated significant up-regulation of IL2 expression-modulating miRNAs in schizophrenia. However, it is known that miRNAs can affect multiple targets. For characterization of enriched functions and biological pathways of the studied miRNAs targets, we used a freely available online integrated system called miRsystem43. The analysis resulted in 30 pathways from 4 databases (KEGG46, Biocarta, Reactome, Pathway Interaction Database) significantly enriched with miRNA targets. The majority of these were related to immune/inflammatory system pathways where IL2 is either an effector or a target gene (Table 4). Figure 5 is an example of miR target enriched pathway46.\n\nRed boxes indicate target genes for studied miRNAs. This figure has been reproduced with permission from the KEGG PATHWAY database46.\n\n\nDiscussion\n\nIn this study, we observed decreased levels of IL2 expression in peripheral blood mononuclear cells of schizophrenic patients, paralleled with increased expression of IL2-regulating miRNAs (miR-31, miR-146a, miR-155 and miR-181c). In addition, we demonstrated that carriage of the minor allele for IL2 rs2069778 is associated with increased IL2 expression levels which might suggest either a regulatory role for this SNP or a linkage with other SNPs that can modulate gene expression.\n\nConflicting results on the levels of IL2 have been reported for this disease both in medicated and undedicated patients. The data published by Singh et al.47 and Theodoropoulou et al.48 are in line with our findings. However, Ebrinc et al.49 and Zhang et al.50,51 have found elevated levels of IL2 in their subjects. Furthermore, there are studies reporting no difference in the IL2 levels of schizophrenic patients as compared to controls52. Because expression was studied in different populations, we can speculate that the observed discrepancies could be partially explained by different genetic backgrounds. Moreover, the validity of our results is supported by the detected increase of IL2 expression-modulating miRNAs measured using independent assay techniques. Finally, consistent with IL2’s role in maintenance of Treg cells, their low levels were also reported in schizophrenia53.\n\nThough the studies of miRNA involvement in schizophrenia are a relatively new direction, there are already results implicating miRNA deregulation in the pathogenesis of schizophrenia. miR-137 is the micro-RNA best known for its role in schizophrenia pathogenesis54–56. This micro-RNA is also well known due to a genetic polymorphism (SNP variant) in its gene, which was described as a genetic risk factor for the development of schizophrenia in a European population57–59. There are other recent studies which confirmed the role of distinct micro-RNAs such as miR-195, miR-181b, miR-301a, miR-19, miR-206, miR-30a and miR-219 in the pathogenesis of schizophrenia60–64. In this study, we reported four miRNAs (miR-31, miR-146a, miR-155, miR-181c) that were up-regulated in schizophrenic patients. Besides targeting IL2 expression, these molecules have many other targets that are involved in immune/inflammatory pathways, confirming the essential role of immune system disturbances in disease development and progression.\n\nThe limitation of the present study is the inability to recruit medication-free patients for assessment of the effect of treatment on IL2 and miRNA expression. However, in many studies cited in this paper50,51, regardless the direction of difference in IL2 levels, no differences were observed between treated and untreated patient groups. It should also be noted that we measured IL2 gene and miRNA expression in two different patient groups with little overlap, which prevented us from performing direct correlation analysis between the levels of IL2 and miRNAs.\n\nOverall, our findings further strengthen the role of immune system deregulation in the development and progression of schizophrenia and necessitate further research towards understanding the changes of the Th1/Th2/T-reg response in this disease and in response to antipsychotic treatment.\n\n\nConclusions\n\nAll studied miRNAs (miR-31, miR-146a, miR-155, miR-181c) were over-expressed in schizophrenic patients, suggesting a role for them in disease pathogenesis. IL2 gene expression was down-regulated in schizophrenic patients. The IL2 rs2069778 C/T SNP is not associated with schizophrenia but regulates expression of the IL2 gene. Abnormalities in studied miRNA expressions result in the deregulation of the T-Cell receptor signaling pathway in schizophrenia.\n\n\nData availability\n\nFigshare: F1000 1990 Ghazaryan et al..xlsx. https://doi.org/10.6084/m9.figshare.9816860.v144.\n\nFigshare: F1000 1990 Ghazaryan et al. Raw Data miRNA.xlsx. https://doi.org/10.6084/m9.figshare.10012442.v345.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe thank the administration and medical staff of the Psychiatric Medical Centers of the Ministry of Health of the Republic of Armenia for selection of patients and healthy subjects.\n\n\nReferences\n\nFaludi G, Dome P, Lazary J: Origins and perspectives of schizophrenia research. Neuropsychopharmacol Hung. 2011; 13(4): 185–192. PubMed Abstract\n\nChase KA, Rosen C, Gin H, et al.: Metabolic and inflammatory genes in schizophrenia. Psychiatry Res. 2015; 225(1–2): 208–211. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFan X, Goff DC, Henderson DC: Inflammation and schizophrenia. Expert Rev Neurother. 2007; 7(7): 789–796. PubMed Abstract \| Publisher Full Text\n\nWatkins CC, Andrews SR: Clinical studies of neuroinflammatory mechanisms in schizophrenia. Schizophr Res. 2016; 176(1): 14–22. PubMed Abstract \| Publisher Full Text\n\nPalomino DC, Marti LC: Chemokines and immunity. Einstein (Sao Paulo). 2015; 13(3): 469–473. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLung FW, Yang MC, Shu BC: The interleukin 10 promoter haplotype ACA and the long-form variant of the DRD4 uVNTR polymorphism are associated with vulnerability to schizophrenia. Psychiatry Res. 2011; 188(2): 294–296. PubMed Abstract \| Publisher Full Text\n\nMisiak B, Stanczykiewicz B, Kotowicz K, et al.: Cytokines and C-reactive protein alterations with respect to cognitive impairment in schizophrenia and bipolar disorder: A systematic review. Schizophr Res. 2018; 192: 16–29. PubMed Abstract \| Publisher Full Text\n\nPotvin S, Stip E, Sepehry AA, et al.: Inflammatory cytokine alterations in schizophrenia: a systematic quantitative review. Biol Psychiatry. 2008; 63(8): 801–808. PubMed Abstract \| Publisher Full Text\n\nZakharyan R, Boyajyan A: Inflammatory cytokine network in schizophrenia. World J Biol Psychiatry. 2014; 15(3): 174–187. PubMed Abstract \| Publisher Full Text\n\nBachmann MF, Oxenius A: Interleukin 2: from immunostimulation to immunoregulation and back again. EMBO Rep. 2007; 8(12): 1142–1148. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde la Rosa M, Rutz S, Dorninger H, et al.: Interleukin-2 is essential for CD4+CD25+ regulatory T cell function. Eur J Immunol. 2004; 34(9): 2480–2488. PubMed Abstract \| Publisher Full Text\n\nDahan S, Bragazzi NL, Yogev A, et al.: The relationship between serum cytokine levels and degree of psychosis in patients with schizophrenia. Psychiatry Res. 2018; 268: 467–472. PubMed Abstract \| Publisher Full Text\n\nGhazaryan H, Petrek M, Boyajyan A: Chronic schizophrenia is associated with over-expression of the interleukin-2 receptor gamma gene. Psychiatry Res. 2014; 217(3): 158–162. PubMed Abstract \| Publisher Full Text\n\nIgue R, Potvin S, Bah R, et al.: Soluble interleukin-2 receptor levels correlated with positive symptoms during quetiapine treatment in schizophrenia-spectrum disorders. Prog Neuropsychopharmacol Biol Psychiatry. 2011; 35(7): 1695–1698. PubMed Abstract \| Publisher Full Text\n\nMahendran R, Mahendran R, Chan YH: Interleukin-2 levels in chronic schizophrenia patients. Ann Acad Med Singapore. 2004; 33(3): 320–323. PubMed Abstract\n\nRapaport MH, McAllister CG, Kim YS, et al.: Increased serum soluble interleukin-2 receptors in Caucasian and Korean schizophrenic patients. Biol Psychiatry. 1994; 35(10): 767–771. PubMed Abstract \| Publisher Full Text\n\nAsevedo E, Rizzo LB, Gadelha A, et al.: Peripheral interleukin-2 level is associated with negative symptoms and cognitive performance in schizophrenia. Physiol Behav. 2014; 129: 194–198. PubMed Abstract \| Publisher Full Text\n\nTan Y, Li Y, Tan S, et al.: Increased interleukin-2 serum levels were associated with psychopathological symptoms and cognitive deficits in treatment-resistant schizophrenia. Schizophr Res. 2015; 169(1–3): 16–21. PubMed Abstract \| Publisher Full Text\n\nKim YK, Kim L, Lee MS: Relationships between interleukins, neurotransmitters and psychopathology in drug-free male schizophrenics. Schizophr Res. 2000; 44(3): 165–175. PubMed Abstract \| Publisher Full Text\n\nPaul-Samojedny M, Owczarek A, Kowalczyk M, et al.: Association of interleukin 2 (IL-2), interleukin 6 (IL-6), and TNF-alpha (TNFα) gene polymorphisms with paranoid schizophrenia in a Polish population. J Neuropsychiatry Clin Neurosci. 2013; 25(1): 72–82. PubMed Abstract \| Publisher Full Text\n\nSchwarz MJ, Krönig H, Riedel M, et al.: IL-2 and IL-4 polymorphisms as candidate genes in schizophrenia. Eur Arch Psychiatry Clin Neurosci. 2006; 256(2): 72–76. PubMed Abstract \| Publisher Full Text\n\nGaffen SL, Liu KD: Overview of interleukin-2 function, production and clinical applications. Cytokine. 2004; 28(3): 109–123. PubMed Abstract \| Publisher Full Text\n\nChen CY, Del Gatto-Konczak F, Wu Z, et al.: Stabilization of interleukin-2 mRNA by the c-Jun NH2-terminal kinase pathway. Science. 1998; 280(5371): 1945–1949. PubMed Abstract \| Publisher Full Text\n\nRanji N, Sadeghizadeh M, Karimipoor M, et al.: MicroRNAs Signature in IL-2-Induced CD4+ T Cells and Their Potential Targets. Biochem Genet. 2015; 53(7–8): 169–183. PubMed Abstract \| Publisher Full Text\n\nKishore A, Borucka J, Petrkova J, et al.: Novel insights into miRNA in lung and heart inflammatory diseases. Mediators Inflamm. 2014; 2014: 259131. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKloosterman WP, Plasterk RH: The diverse functions of microRNAs in animal development and disease. Dev Cell. 2006; 11(4): 441–450. PubMed Abstract \| Publisher Full Text\n\nTomankova T, Petrek M, Gallo J, et al.: MicroRNAs: Emerging Regulators of Immune-Mediated Diseases. Scand J Immunol. 2012; 75(2): 129–141. PubMed Abstract \| Publisher Full Text\n\nJiang Q, Wang Y, Hao Y, et al.: miR2Disease: a manually curated database for microRNA deregulation in human disease. Nucleic Acids Res. 2009; 37(Database issue): D98–104. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeveridge NJ, Gardiner E, Carroll AP, et al.: Schizophrenia is associated with an increase in cortical microRNA biogenesis. Mol Psychiatry. 2010; 15(12): 1176–1189. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFeng J, Sun G, Yan J, et al.: Evidence for X-chromosomal schizophrenia associated with microRNA alterations. PLoS One. 2009; 4(7): e6121. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHommers LG, Domschke K, Deckert J: Heterogeneity and individuality: microRNAs in mental disorders. J Neural Transm (Vienna). 2015; 122(1): 79–97. PubMed Abstract \| Publisher Full Text\n\nMoreau MP, Bruse SE, David-Rus R, et al.: Altered microRNA expression profiles in postmortem brain samples from individuals with schizophrenia and bipolar disorder. Biol Psychiatry. 2011; 69(2): 188–193. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCurtale G, Citarella F, Carissimi C, et al.: An emerging player in the adaptive immune response: microRNA-146a is a modulator of IL-2 expression and activation-induced cell death in T lymphocytes. Blood. 2010; 115(2): 265–273. PubMed Abstract \| Publisher Full Text\n\nFan W, Liang D, Tang Y, et al.: Identification of microRNA-31 as a novel regulator contributing to impaired interleukin-2 production in T cells from patients with systemic lupus erythematosus. Arthritis Rheum. 2012; 64(11): 3715–3725. PubMed Abstract \| Publisher Full Text\n\nXue Q, Guo ZY, Li W, et al.: Human activated CD4+ T lymphocytes increase IL-2 expression by downregulating microRNA-181c. Mol Immunol. 2011; 48(4): 592–599. PubMed Abstract \| Publisher Full Text\n\nMao CP, He L, Tsai YC, et al.: In vivo microRNA-155 expression influences antigen-specific T cell-mediated immune responses generated by DNA vaccination. Cell Biosci. 2011; 1(1): 3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBouzid D, Fourati H, Amouri A, et al.: Autoimmune diseases association study with the KIAA1109-IL2-IL21 region in a Tunisian population. Mol Biol Rep. 2014; 41(11): 7133–7139. PubMed Abstract \| Publisher Full Text\n\nFirst MB: Diagnostic and statistical manual of mental disorders, 5th edition, and clinical utility. J Nerv Ment Dis. 2013; 201(9): 727–729. PubMed Abstract \| Publisher Full Text\n\nMiller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988; 16(3): 1215. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWelsh K, Bunce M: Molecular typing for the MHC with PCR-SSP. Rev Immunogenet. 1999; 1(2): 157–176. PubMed Abstract\n\nMcDermott AM, Kerin MJ, Miller N: Identification and validation of miRNAs as endogenous controls for RQ-PCR in blood specimens for breast cancer studies. PLoS One. 2013; 8(12): e83718. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBunce M, Fanning GC, Welsh KI: Comprehensive, serologically equivalent DNA typing for HLA-B by PCR using sequence-specific primers (PCR-SSP). Tissue Antigens. 1995; 45(2): 81–90. PubMed Abstract \| Publisher Full Text\n\nLu TP, Lee CY, Tsai MH, et al.: miRSystem: an integrated system for characterizing enriched functions and pathways of microRNA targets. PLoS One. 2012; 7(8): e42390. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGhazaryan H: F1000 1990 Ghazaryan et. al..xlsx. 2019. http://www.doi.org/10.6084/m9.figshare.9816860.v1\n\nGhazaryan H: F1000 1990 Ghazaryan et al Raw Data miRNA.xlsx. 2019. http://www.doi.org/10.6084/m9.figshare.10012442.v3\n\nKanehisa M, Goto S: KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000; 28(1): 27–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSingh B, Bera NK, Nayak CR, et al.: Decreased serum levels of interleukin-2 and interleukin-6 in Indian Bengalee schizophrenic patients. Cytokine. 2009; 47(1): 1–5. PubMed Abstract \| Publisher Full Text\n\nTheodoropoulou S, Spanakos G, Baxevanis CN, et al.: Cytokine serum levels, autologous mixed lymphocyte reaction and surface marker analysis in never medicated and chronically medicated schizophrenic patients. Schizophr Res. 2001; 47(1): 13–25. PubMed Abstract \| Publisher Full Text\n\nEbrinc S, Top C, Oncul O, et al.: Serum interleukin 1 alpha and interleukin 2 levels in patients with schizophrenia. J Int Med Res. 2002; 30(3): 314–317. PubMed Abstract \| Publisher Full Text\n\nZhang XY, Zhou DF, Cao LY, et al.: Cortisol and cytokines in chronic and treatment-resistant patients with schizophrenia: association with psychopathology and response to antipsychotics. Neuropsychopharmacology. 2005; 30(8): 1532–1538. PubMed Abstract \| Publisher Full Text\n\nZhang XY, Zhou DF, Zhang PY, et al.: Elevated interleukin-2, interleukin-6 and interleukin-8 serum levels in neuroleptic-free schizophrenia: association with psychopathology. Schizophr Res. 2002; 57(2–3): 247–258. PubMed Abstract \| Publisher Full Text\n\nFreudenreich O, Brockman MA, Henderson DC, et al.: Analysis of peripheral immune activation in schizophrenia using quantitative reverse-transcription polymerase chain reaction (RT-PCR). Psychiatry Res. 2010; 176(2–3): 99–102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFernandez-Egea E, Vertes PE, Flint SM, et al.: Peripheral Immune Cell Populations Associated with Cognitive Deficits and Negative Symptoms of Treatment-Resistant Schizophrenia. PLoS One. 2016; 11(5): e0155631. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCollins AL, Kim Y, Bloom RJ, et al.: Transcriptional targets of the schizophrenia risk gene MIR137. Transl Psychiatry. 2014; 4: e404. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOlde Loohuis NF, Nadif Kasri N, Glennon JC, et al.: The schizophrenia risk gene MIR137 acts as a hippocampal gene network node orchestrating the expression of genes relevant to nervous system development and function. Prog Neuropsychopharmacol Biol Psychiatry. 2017; 73: 109–118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWhalley HC, Papmeyer M, Romaniuk L, et al.: Impact of a microRNA MIR137 susceptibility variant on brain function in people at high genetic risk of schizophrenia or bipolar disorder. Neuropsychopharmacology. 2012; 37(12): 2720–2729. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFatima A, Farooq M, Abdullah U, et al.: Genome-Wide Supported Risk Variants in MIR137, CACNA1C, CSMD1, DRD2, and GRM3 Contribute to Schizophrenia Susceptibility in Pakistani Population. Psychiatry Investig. 2017; 14(5): 687–692. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGonzález-Giraldo Y, González-Reyes RE, Forero DA: A functional variant in MIR137, a candidate gene for schizophrenia, affects Stroop test performance in young adults. Psychiatry Res. 2016; 236: 202–205. PubMed Abstract \| Publisher Full Text\n\nWarburton A, Breen G, Bubb VJ, et al.: A GWAS SNP for Schizophrenia Is Linked to the Internal MIR137 Promoter and Supports Differential Allele-Specific Expression. Schizophr Bull. 2016; 42(4): 1003–1008. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlacam H, Akgun S, Akca H, et al.: miR-181b-5p, miR-195-5p and miR-301a-3p are related with treatment resistance in schizophrenia. Psychiatry Res. 2016; 245: 200–206. PubMed Abstract \| Publisher Full Text\n\nHan J, Gage FH: A role for miR-19 in the migration of adult-born neurons and schizophrenia. Neurogenesis (Austin). 2016; 3(1): e1251873. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHauberg ME, Holm-Nielsen MH, Mattheisen M, et al.: Schizophrenia risk variants affecting microRNA function and site-specific regulation of NT5C2 by miR-206. Eur Neuropsychopharmacol. 2016; 26(9): 1522–1526. PubMed Abstract \| Publisher Full Text\n\nLiu S, Zhang F, Shugart YY, et al.: The early growth response protein 1-miR-30a-5p-neurogenic differentiation factor 1 axis as a novel biomarker for schizophrenia diagnosis and treatment monitoring. Transl Psychiatry. 2017; 7(1): e998. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMurai K, Sun G, Ye P, et al.: The TLX-miR-219 cascade regulates neural stem cell proliferation in neurodevelopment and schizophrenia iPSC model. Nat Commun. 2016; 7: 10965. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "62639", "date": "20 May 2020", "name": "Mirko Grubor", "expertise": [ "Reviewer Expertise Pharmacogenomics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, authors investigated association of miR-31, miR-146a, miR-181c and miR-155 with schizophrenia pathogenesis, as well as their link to IL2 gene expression in disease. It was noted that 225 patients with paranoid schizophrenia and 225 sex- and age-matched controls were recruited in study. Peripheral blood samples of each patient was used. Results have shown a higher expression of studied mi-RNAs in schizophrenic patients vs control group. As well as lower expression of IL2 gene in schizophrenic patients vs control group. IL2 rs2069778 SNP was not associated with schizophrenia.\nIt is kindly requested from authors to address following comments:\nIn Introduction section, it is indicated that interleukins and other cytokines and their receptors play an essential role schizophrenia predisposition, development, progression and treatment response. Most of the cited literature reported non-conclusive results in order to support such strong statement. Consideration to rephrase wording \"essential\" is suggested.\n\nIt is stated that 225 patients with paranoid schizophrenia and 225 sex- and age-matched controls were recruited in study yet only 61 and 66 patients were tested for micro-RNA expression and IL2 gene expression respectively. It is not clear which inclusion and exclusion criteria were utilized to have only 127 patients tested in total. Providing more information/explanation on these criteria is suggested. It is also stated that all subjects were genotyped for this study. Does this refer to total number of patients or only to ones which were tested or micro-RNA expression and IL2 gene expression?\n\nNo information on disease stage, type of medications, relevant medical history, concomitant medications and especially comorbidity was provided/discussed for tested patients. MiRNA and IL2 can effectively participate in the pathogenesis of several pathological conditions, such as cancer and metabolic, infectious, autoimmune and inflammatory diseases, Possibility of such comorbidity and influence on the study results should be considered and discussed.\n\nAlso, observed results of differences in miR-181c and miR-31 expression in T allele carriers should be discussed in more detail.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "65949", "date": "21 Jul 2020", "name": "Ghanshyam N. Pandey", "expertise": [ "Reviewer Expertise Neurology of mood disorders", "schizophrenia and suicide." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study the authors determined several micro-RNAs and mRNA expression of IL-2 in the PBMC obtained from 225 paranoid schizophrenic patients and 225 healthy normal control subjects. They found that all studied micro-RNAs, for example miR-16, miR31, miR-146a, miR-155 and miR-181c were overexpressed in the peripheral blood mononuclear cells (PBMC) of schizophrenic subjects compared with normal control subjects. They also found that the IL-2 mRNA expression was down regulated in schizophrenic patients compared with normal control subjects.\nThis is an important study of miRNA and IL-2 in schizophrenia. The results are significant.\nThere are many strong points of the study. The number of study subjects is very large -- 225 patients with paranoid schizophrenia and 225 sex- and age-matched normal control subjects. They determined relevant miRNAs in the schizophrenic patients. They also determined the mRNA expression for IL-2, which has been studied in schizophrenia and was found down-regulated in this study. A procedure for isolating the RNA from PBMC is well described and the methods used for mRNA determinations and miRNA determination are also sound. The Introduction is to the point and relevant. The Discussion is pertinent and the Methods are described clearly. Overall, this is a very important and strong study of miRNA and IL-2 in schizophrenia.\n\nI have only a few minor comments:\nWhat was the drug-status of the schizophrenic patients at the time of this study? Were all of them drug-free or were on treatment with any drugs?\n\nThey studied the mRNA expression of IL-2 in schizophrenic patients. The rationale for studying IL-2 has been described. However, it is not clear why they did not study the mRNA expression of the cytokines IL-1β, IL-6 and TNF-α, the abnormalities of which have been implicated in schizophrenia.\n\nThey determined the mRNA expression of IL-2 and were comparing the results with the protein expression determined by other investigators in the plasma of schizophrenic patients. These comparisons may be fine but may not be relevant. Can they describe if there are other studies of mRNA in schizophrenic patients and maybe they should cite those. For example, Pandey et al. (2015, 20181,2).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-2077
https://f1000research.com/articles/8-2073/v1	10 Dec 19	{ "type": "Software Tool Article", "title": "Cancer Publication Portal: an online tool for summarizing and searching human cancer-genomic publications", "authors": [ "Garrett M. Dancik", "Kevin Williams", "Myron Zhang", "Nataliia Romanenko", "Kevin Williams", "Myron Zhang", "Nataliia Romanenko" ], "abstract": "A search of PubMed lists >582,000 citations with the keywords “cancer” and “gene”. The large volume of cancer genomic publications necessitates the development of text-mining tools to help cancer researchers navigate and summarize articles efficiently. We developed a Cancer Publication Portal (CPP) to help researchers efficiently search and summarize cancer genomic publications, based on one or more genes of interest. CPP integrates data from several sources, including PubTator, the Medical Subject Headings (MeSH) database; the HUGO Gene Nomenclature Committee human gene name database; PubMed, a database of biomedical literature citations; and the National Cancer Institute (NCI) Thesaurus. Following each query, results are summarized and include the publication frequency for each cancer type, as well as publication frequencies for cancer terms, pharmacological agents, genomic mutations, and additional genes stratified by cancer type. Cancer terms were identified by comparing titles and abstracts from cancer-related (N=851,868) and non-cancer related articles (N=2,607,020). CPP allows a user to quickly obtain publication statistics, such as the frequency of articles mentioning EGFR across cancer types, and to explore associations, such as the association between pharmacological agent and cancer type. Result summaries are interactive, so additional filters can be easily added as the literature is explored. After a search is completed, a PubTator collection can be quickly created, in order to view article titles and abstracts in PubTator. CPP currently includes information for ~1.1 million cancer-related publications associated with >23,000 human genes. Database URL: https://gdancik.github.io/bioinformatics/CPP/.", "keywords": [ "Text-mining", "Cancer" ], "content": "Introduction\n\nCancer is a genetic disease1, with relevant genes often identified through functional screening2–4, gene expression profiling5–7, or genomic sequencing experiments8–10. While researchers may need to quickly understand the published literature regarding a particular gene, the large volume of publications can make this challenging. Indeed, a search of PubMed finds >582,000 citations with the keywords “cancer” and “gene”, with >175,000 articles published within the previous 5 years. The large volume of cancer genomic publications necessitates the development of tools to help cancer researchers navigate and summarize articles efficiently.\n\nThe use of controlled vocabularies and text-mining tools has facilitated the annotation and searching of biomedical literature. In particular, the National Library of Medicine’s Medical Subject Headings provides a controlled vocabulary of MeSH terms for indexing MEDLINE/PubMed articles. PubTator is a web-based platform, designed to assist biocuration, that uses robust text-mining tools to annotate PubMed articles with respect to genes, chemicals, species, and mutations11. However, while PubTator allows users to search PubMed based on these biological concepts, summaries of the results are not provided. Other tools such as Anne O’Tate12 and PubReminer13 summarize PubMed searches, but are not cancer-specific and have limitations regarding the number of results that can be returned. Anne O’Tate, for example, allows users to search PubMed and summarizes the results based on important words, phrases, authors, and other fields12. PubReminer13 also allows PubMed queries and summarizes articles based on common words, MeSH terms, and other fields. These summaries are useful but are not cancer specific, and cancer type mentions can be difficult to find and may not appear in the search results.\n\nHere we develop a Cancer Publication Portal (CPP), a web application to help users search and summarize the cancer genomic literature14,15. CPP allows a user to enter a human gene or gene set of interest, and then summarizes the relevant cancer-related publications mentioning this gene through tabular and graphical summaries showing the frequency of articles by cancer type, pharmacological agent, genomic mutation, and additional human genes mentioned in article titles or abstracts. Additionally, CPP catalogs and summarizes articles based on mentions of >30 cancer-related terms. The tool is designed to provide users with publication statistics, such as the number of articles mentioning EGFR and erlotinib across cancer types, as well as to facilitate exploration and retrieval of relevant articles. For example, a user can quickly find all articles based on a set of genes and a collection of cancer types. Interactive summaries allow users to narrow in on a topic of interest by applying additional filters. Results are summarized across cancer types, and the process can be repeated. At any point, the user can access article abstracts from PubTator, as well as download statistical summaries. In this fashion, researchers and students can explore the literature and find articles in a gene- and cancer-focused way.\n\n\nMethods\n\nCPP14,15 integrates data from a variety of sources including PubTator11, the Medical Subject Headings (MeSH) database; the HUGO Gene Nomenclature Committee (HGNC) human gene name database16, the PubMed database of biomedical literature citations, and the National Cancer Institute (NCI) Thesaurus17. An overview of data collection is provided in Figure 1A. Generally, article association data for genes, chemicals, diseases, and mutations was collected from PubTator, then updated and filtered as described below and in Figure 1A. Cancer term associations were identified by comparing PubMed abstracts, as described below and in Figure 1A. Summary statistics for CPP, which contains article-entity association information for 1,143,191 articles and 19,551 genes, is provided in Table 1. The mean number of articles per gene is 138.6 (median = 9.0), but the number of publications per gene is uneven, with the 192 most frequently mentioned genes accounting for >50% of all publications. The three most frequently mentioned genes are TNF (N = 64,103), TP53 (62,602), and EGFR (35,178) (Extended data, Supplementary Table S1)18. The three most common cancer types are Breast Neoplasms (N = 119,891), Leukemia (N = 103,222), and Lymphoma (N = 60,320) (Extended data, Supplementary Table S2)18.\n\n(A) CPP integrates data from PubTator, PubMed, HGNC, MeSH, and the NCI Thesaurus to summarize articles based on their references to cancer type, mutations, genes, and cancer-related terms. (B) Selected cancer-related terms identified from the titles/abstracts of ~3.5 million publications. The log10 ratio of the cancer publication frequency to non-cancer publication frequency is shown.\n\nCollection and processing of PubTator data. Data defining article-gene, article-disease, article-chemical, and article-mutation relationships were downloaded from PubTator via FTP. PubTator data defines associations between articles and mentions of genes, chemicals, diseases, and mutations in article titles or abstracts. MeSH terms for descriptor and supplemental record sets were downloaded as XML files and parsed to extract MeSH IDs and their corresponding terms. A list of pharmacologically active compounds was also downloaded from MeSH via its FTP service. PubTator data was filtered to include only those articles mentioning human genes and only those articles that are cancer-related, i.e., that mention MeSH terms falling under the heading “Neoplasm” (Tree Number C04). We take advantage of MeSH tree structure and remove redundant MeSH IDs if a child MeSH ID is mentioned in the same article. For example, an article mentioning both “Breast Neoplams” (C04.588.180) and “Triple Negative Breast Neoplasms” (C04.588.180.788) would have the former removed. We also recode “Neoplasms” (C04) as “Neoplasms (unspecified)” (C04.000) if this is the only cancer MeSH term associated with the article. Obsolete MeSH IDs were updated by testing the terms associated with that entry against current MeSH headings and Supplementary Concept record terms. Mutation data was reformatted according to HGVS sequence variant nomenclature recommendations19. For each mutation, we identify the gene or genes most commonly associated with it, and store this information in CPP.\n\nIdentification and annotation of cancer terms. In addition to the article associations provided by PubTator, we report associations between articles and “cancer terms”. We identified cancer-terms by comparing title/abstract word ‘stems’ between cancer-related (N=851,868) and non-cancer related articles (N=2,607,020) that mentioned at least one human gene. Abstracts were downloaded from PubMed’s FTP service. These titles/abstracts contained a total of 5,564 unique word stems, with 2,633 word stems more common in cancer articles (P < 0.01, Fisher’s exact test). In order to focus on word stems that would be most informative in a cancer-specific context, we filtered these results by considering only word stems that occurred in > 1% of cancer-related articles. Word stems related to disease/tissue (e.g., ‘renal’), gene name (e.g., ‘kinase’), and miscellaneous words (e.g., ‘report’) were also filtered out. Word stems for similar words were combined, based on common word usage and the NCI Thesaurus17. In addition, several terms deemed important by the authors (e.g., “immunotherapy”) were added, even if occurring < 1% of the time in cancer-related articles. The full list of 37 cancer terms can be seen in the Extended data, Supplementary Table S318. Selected terms are shown in Figure 1B.\n\nFor each cancer term, we find a non-redundant set of word stems corresponding to the term itself and its synonyms according to the NCI thesaurus (Extended data, Supplementary Table S3)18. Such an approach allows us to identify a concept (e.g., ‘mutation’) even when a synonym (e.g., ‘genetic alteration’) is used. For each cancer-related article, we search its title/abstract for mentions of cancer terms and add these relationships to the CPP database.\n\nTechnical details. Python and R v3.5.2 were used for data processing. PubMed files were parsed using the Python “PubMed Parser”20 and word stems found using the Snowball stemmer from Python’s NLTK module, after removal of stop words and any word with no more than 3 characters. Additional XML files were parsed using the Python module lxml. After processing, data was loaded into a MySQL database. The web interface was developed using R/Shiny v1.2.0.\n\nCPP runs in a standard web browser and is available for public use at the following address: https://gdancik.github.io/bioinformatics/CPP/.\n\n\nUse cases\n\nCPP14,15 takes a gene-centric approach for finding and summarizing cancer-related articles based on mentions of cancer types, cancer terms, drugs, mutations, and additional genes. In order to demonstrate the utility of CPP, we use CPP to summarize and explore cancer-related articles mentioning the gene epidermal growth factor receptor (EGFR), a gene mutated in >30% of patients with non-small cell lung cancer21 and a gene that can be targeted by tyrosine kinase inhibitors such as gefitinib and erlotinib22. The user starts by selecting the desired gene from the drop-down menu. After selecting EGFR, CPP tells us that there are 35,178 articles found, covering 422 cancer types (Figure 2A). We note that “cancer types” here is defined according to MeSH subject headings, which categorize cancers by both site and histological type. The top three cancer types are “Neoplasms, Glandular and Epithelial”, “Thoracic Neoplasms”, and “Lung Neoplasms”. We next select the cancer types to search, by either clicking on the table or selecting cancer types from the drop-down menu. Cancer types can also be uploaded from a file. After clicking on the button to retrieve the summaries, we get a tabular and graphical summary showing the number of articles mentioning both the selected gene and each selected cancer type (Figure 2B). If no cancer type is selected, then all cancer types will be summarized. Here it is easy to compare the number of articles mentioning EGFR across cancer types, and a user quickly sees that lung cancer is the most common.\n\n(A) Cancer selection screen displayed after a user enters one or more genes. (B) Cancer types summary screen, showing frequency table and bar graph of selected cancer types.\n\nFigure 3 shows additional summaries provided by CPP. Summaries include frequency tables showing the number of articles associated with the selected gene and Cancer Terms, Drugs, Mutations, and Additional Genes; and stacked bar graphs for visualizing the distribution of each entity across cancer types. If the user searched for multiple genes, a summary of the selected genes is also provided. The frequency tables allow a user to quickly identify entities (such as drugs) that are commonly mentioned in the literature, while the stacked bar graphs allow a user to qualitatively evaluate when entities are more (or less) associated with specific cancer types than others. In this example, frequency tables show that gefitinib and erlotinib are the two drugs most commonly associated with EGFR (Figure 3A), the EGFR mutations p.T790M and p.L858R are most common (Figure 3B), and ERBB2, KRAS, and TP53 are the genes that most frequently co-occur with EGFR (Figure 3C). However, in the latter case the stacked bar graph shows that these co-occurring genes are associated with specific cancer types. Specifically, while KRAS is the most common gene that co-occurs with EGFR in lung cancer, the most common genes that co-occur with breast cancer and glioma are ERBB2 and TP53, respectively (Figure 3C). Such associations may reflect genomic differences between cancer types, or may reflect a bias in the literature. The stacked bar graphs are interactive. A user can single click on an entity in the legend to hide the entity from the graph, and double-click on an entity to hide all other entities. This toggling can be canceled by clicking or double clicking the entity a second time. For example, by double clicking on the drug irinotecan, we can see that this drug is associated with colorectal cancers more than other cancer types (Figure 3A, inset).\n\nSummaries and stacked bar graphs are provided for Cancer terms (not shown), (A) Drugs, (B) Mutations, and (C) mentions of additional Genes. Inset in (A) shows only irinotecan, obtained by double clicking on that drug in the legend.\n\nIn addition to summarizing the cancer genomic literature, CPP is designed to help users explore the literature and quickly find articles of interest. After selecting one or more genes and cancer types, a user can add filters by clicking on one or more rows of any frequency table to add an entity to the filter. For each entity type, the user can retrieve articles that mention either all of the selected terms or any of the selected terms. For example, a user interested in publications assessing the predictive value of EGFR mutations as biomarkers for gefitinib or erlotinib treatment could use CPP to first find articles that mention EGFR and any cancer type. Then the user can specify additional filters to limit the results to articles that mention both mutation and survival, and either of the drugs gefitinib or erlotinib (Figure 4A). Note that cancer term filters are based on word ‘stems’ and synonyms from the NCI Thesaurus, and therefore will recognize variations of the search term. For example, the cancer term “mutation” includes any words with a stem of ‘mutat’, which includes the words “mutation”, “mutations”, and “mutated”; and word stems corresponding to ‘genetic alteration’ and ‘genetic change’ (Extended data, Supplementary Table S3)18. This search results in 1,169 articles being found. In summarizing the articles, we see that lung cancers are the dominant cancer type (Figure 4B), and that the number of articles is similar for each drug across cancer types (Figure 4C). We note that the stacked bar graphs are now limited to the entities we have selected (i.e., gefitinib and erlotinib).\n\n(A) Filters can be specified for all selected terms for an entity or any selected term. Current filter shows articles mentioning mutation and survival and either gefitinib or erlotinib. (B) Cancer summary of results for EGFR, all cancer types, and the filters in (A). (C) Stacked bar graph showing mentions of gefitinib and erlotinib across cancer types. (D) Screenshot of ‘Articles’ tab where user can create a PubTator collection to view the current set of articles.\n\nUsers can easily explore the literature by adding and removing filters. At any point, a user can view abstracts for the current set of articles. A user views abstracts by selecting the ‘Articles’ tab, clicking the ‘Copy PMIDs’ button, and then creating a new collection in PubTator, which is displayed on the Articles page using an iframe (Figure 4D). This allows a user to seamlessly view relevant abstracts after applying the desired filters. Additionally, a user can download results to a CSV file, for the current list of PMIDs, as well as frequency summaries for Cancer Types, Cancer Terms, Drugs, Mutations, and Additional Genes from the ‘Download’ tab.\n\n\nDiscussion and conclusions\n\nCPP14,15 is designed to help users efficiently explore and summarize the cancer genomic literature, and should be useful for cancer researchers who are looking for relevant articles for a gene of interest, for meta-researchers who study the publication landscape, and for students learning about the relationship between one or more genes and cancer types. Because CPP quickly summarizes articles across cancer types, CPP can be used to assess whether a gene might be novel for a particular cancer type (or any cancer type), based on the frequency of gene mentions in titles/abstracts of cancer-related publications. CPP also provides summaries of cancer terms, drugs, mutations, and co-occurring genes that can connect a researcher to key biological concepts and the underlying articles that might inform their research. The use of cancer terms, unique to CPP, summarizes articles in a cancer-specific way and allows for quick retrieval of articles based on a cancer term, such as tumor suppressor, chemotherapy, or metastasis. Furthermore, because CPP identifies associations between articles and cancer terms by using the NCI Thesaurus and ‘stem’ words to cover synonyms and variant word forms (such as ‘mutation’ and ‘mutated’), CPP will retrieve a more valid set of articles than a simple PubMed search for a term in other databases.\n\nDespite its utility, CPP has several limitations that are common in all text-mining based tools. With the exception of cancer terms, article associations in CPP are derived from PubTator associations. While some associations may be missed, PubTator uses cutting edge text-mining tools and the F1 scores for gene, disease, and mutation identification are all > 80%11.\n\nImportantly, text-mining associations are determined only by textual relationships and may not reflect underlying biology. For example, genes that are mentioned together in the same abstract may or may not interact. Similarly, publication frequency may reflect publication bias and not biological importance. Other tools are available for looking at specific biological relationships, such as STRING for protein interactions23, and cBioPortal for Cancer Genomics for exploring genomic datasets24. CPP is designed to complement these tools by providing an overview of the cancer genomic literature and by helping researchers quickly find relevant publications. Finally, all CPP associations, which are derived from PubTator, are based on entity mentions in titles and abstracts only. However, the recently released PubTator Central includes associations from the PubMed Central (PMC) Text Mining Subset25. PMC contains the full text of ~3 million articles, though we expect <4% to be cancer-related. PubTator Central associations will be integrated into CPP in a future release.\n\nIn conclusion, CPP is an easy-to-use web application that allows researchers to efficiently summarize and search the cancer literature for articles based on one or more genes of interest. CPP will be updated approximately once a month following PubTator data releases.\n\n\nData availability\n\nAssociations between genes, diseases, chemicals, mutations, and articles were downloaded from the PubTator FTP page (ftp://ftp.ncbi.nlm.nih.gov/pub/lu/PubTator/)\n\nMeSH descriptor files were downloaded from https://www.nlm.nih.gov/databases/download/mesh.html\n\nDataverse: Cancer Publication Portal: an online tool for summarizing and searching human cancer-genomic publications (supporting data). https://doi.org/10.7910/DVN/BYKF1L18\n\nThis project contains the following extended data:\n\nSupplementary Table S1. Number of articles per gene in Cancer Publication Portal.\n\nSupplementary Table S2. Number of articles per cancer type in Cancer Publication Portal. Cancer types are defined by cancer-related MeSH TreeIDs (C04).\n\nSupplementary Table S3. Cancer terms and synonyms included in Cancer Publication Portal. For each term, there was a statistically significant difference in the proportion of mentions between cancer-related and non-cancer related articles (P < 0.001 by Fisher’s exact test). , term is included despite appearing in < 1% of cancer-related articles, and/or not being cancer-specific (i.e., log10 ratio < 1).\n\nExtended data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nSoftware availability\n\nCPP is available as a web resource at: https://gdancik.github.io/bioinformatics/CPP/.\n\nSource code for the web interface is available from: https://github.com/gdancik/CPP.\n\nSource code for data processing and database creation is available from: https://github.com/gdancik/CPP_setup.\n\nThe database is available from the following docker image: https://hub.docker.com/r/gdancik/dcast.\n\nArchived source code for web interface at time of publication: https://doi.org/10.5281/zenodo.355011014.\n\nArchived source for data processing and database creation at time of publication: https://doi.org/10.5281/zenodo.355011215.\n\nLicense: GNU General Public License-2.\n\n\nAcknowledgements\n\nThe authors acknowledge Stefanos Stravoravdis for coding contributions, and Andrew Johnson for coding contributions and technical assistance. The authors also acknowledge Jason Duex and Sunny Guin for testing and providing feedback for an earlier version of the tool.", "appendix": "References\n\nStratton MR, Campbell PJ, Futreal PA: The cancer genome. Nature. 2009; 458(7239): 719–24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuin S, Pollard C, Ru Y, et al.: Role in tumor growth of a glycogen debranching enzyme lost in glycogen storage disease. J Natl Cancer Inst. 2014; 106(5): pii: dju062. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHan R, Li L, Ugalde AP, et al.: Functional CRISPR screen identifies AP1-associated enhancer regulating FOXF1 to modulate oncogene-induced senescence. Genome Biol. 2018; 19(1): 118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFalkenberg KJ, Newbold A, Gould CM, et al.: A genome scale RNAi screen identifies GLI1 as a novel gene regulating vorinostat sensitivity. Cell Death Differ. 2016; 23(7): 1209–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYouns M, Efferth T, Reichling J, et al.: Gene expression profiling identifies novel key players involved in the cytotoxic effect of Artesunate on pancreatic cancer cells. Biochem Pharmacol. 2009; 78(3): 273–83. PubMed Abstract \| Publisher Full Text\n\nLee RS, Zhang L, Berger A, et al.: Characterization of the ERG-regulated Kinome in Prostate Cancer Identifies TNIK as a Potential Therapeutic Target. Neoplasia. 2019; 21(4): 389–400. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReyes I, Reyes N, Suriano R, et al.: Gene expression profiling identifies potential molecular markers of papillary thyroid carcinoma. Cancer Biomark. 2019; 24(1): 71–83. PubMed Abstract \| Publisher Full Text\n\nCollins CC, Volik SV, Lapuk AV, et al.: Next generation sequencing of prostate cancer from a patient identifies a deficiency of methylthioadenosine phosphorylase, an exploitable tumor target. Mol Cancer Ther. 2012; 11(3): 775–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLabgaa I, Villacorta-Martin C, D'Avola D, et al.: A pilot study of ultra-deep targeted sequencing of plasma DNA identifies driver mutations in hepatocellular carcinoma. Oncogene. 2018; 37(27): 3740–52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee WK, Lee SG, Yim SH, et al.: Whole Exome Sequencing Identifies a Novel Hedgehog-Interacting Protein G516R Mutation in Locally Advanced Papillary Thyroid Cancer. Int J Mol Sci. 2018; 19(10): pii: E2867. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWei CH, Kao HY, Lu Z: PubTator: a Web-based text mining tool for assisting Biocuration. Nucleic Acids Res. 2013; 41(Web Server issue): W518–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmalheiser NR, Zhou W, Torvik VI: Anne O’Tate: A tool to support user-driven summarization, drill-down and browsing of PubMed search results. J Biomed Discov Collab. 2008; 3: 2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPubReMiner: a tool for PubMed query building and literature mining [Internet]. [cited 2019 Jun 17]. Reference Source\n\nDancik G, Johnson A, Romanenko N: gdancik/CPP: CPP (F1000 release) (Version 1.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3550110\n\nWilliams K, Zhang M, Dancik G: gdancik/CPP_setup: CPP_setup (F1000Research) (Version 1.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3550112\n\nYates B, Braschi B, Gray KA, et al.: Genenames.org: the HGNC and VGNC resources in 2017. Nucleic Acids Res. 2017; 45(D1): D619–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNCI Thesaurus [Internet]. [cited 2019 Jun 20]. Reference Source\n\nDancik G, Williams K, Zhang M, et al.: Cancer Publication Portal: an online tool for summarizing and searching human cancer-genomic publications (supporting data). Harvard Dataverse, V1, UNF:6:5POzQ6fu7p4qBw5J6vIFpQ== [fileUNF]. 2019. http://www.doi.org/10.7910/DVN/BYKF1L\n\nden Dunnen JT, Dalgleish R, Maglott DR, et al.: HGVS Recommendations for the Description of Sequence Variants: 2016 Update. Hum Mutat. 2016; 37(6): 564–9. PubMed Abstract \| Publisher Full Text\n\nAchakulvisut T, Acuna DE: PubMed Parser [Internet]. PubMed Parser. [cited 2015 Jul 2]. 2015. Reference Source\n\nZhang YL, Yuan JQ, Wang KF, et al.: The prevalence of EGFR mutation in patients with non-small cell lung cancer: a systematic review and meta-analysis. Oncotarget. 2016; 7(48): 78985–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRocha-Lima CM, Soares HP, Raez LE, et al.: EGFR targeting of solid tumors. Cancer Control. 2007; 14(3): 295–304. PubMed Abstract \| Publisher Full Text\n\nSzklarczyk D, Franceschini A, Wyder S, et al.: STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res. 2015; 43(Database issue): D447–452. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGao J, Aksoy BA, Dogrusoz U, et al.: Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal. 2013; 6(269): pl1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWei CH, Allot A, Leaman R, et al.: PubTator central: automated concept annotation for biomedical full text articles. Nucleic Acids Res. 2019; 47(W1): W587–93. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "58622", "date": "04 Feb 2020", "name": "Elspeth A. Bruford", "expertise": [ "Reviewer Expertise Human genomics", "genetics", "comparative genomics", "bioinformatics", "nomenclature", "biomedical resources." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a new tool that aims to simplify searching for cancer-related publications. There is no doubt that the number of publications in this field is ever increasing and hence a tool like this could prove useful in narrowing down the number of citations that could be of interest.\nIs the description of the software tool technically sound?\nI said \"partly\" as the \"technical details\" within the manuscript itself number only a total of 11 lines of text and don't really give any description as such - what does \"Snowball stemmer\" do? What PubMed files were parsed? Etc.\nHowever, at the end of the manuscript the authors state all of the software is freely available in GitHub, so while there is very little discussion of the software tool itself, hopefully someone could reconstruct the work using the software listed (I wasn't about to try this).\nI used the URL listed for accessing the tool - in fact this URL then directs you to another URL, http://bioinformatics.eaternct.edu/app/CPP/. I have no idea why that URL isn't actually listed in the manuscript, and it was disappointing to see that they haven't bothered to make this https instead of http.\n\nA few comments on the user interface: the home button doesn't seem to take you anywhere - if you've run a search and click home you stay exactly where you are on the results page. Also clicking on \"Cancer Publication Portal\" doesn't take you anywhere; personally I find it quite irritating that the initial stages of any search, selecting your filters, involve a modal popup window, which then closes, it also makes modifying the terms clunky; the order of the genes listed in the drop-down seems very odd (I think it could be based on the NCBI Gene ID for each gene?) - it would make more sense to make the list alphabetical; when selecting the \"terms\", it would be helpful to have an \"X\" next to each selected term to make it clear how they can be removed (clicking delete did remove the term but it also did something odd to the \"selected\" box, where a dropdown suddenly appeared...(using Firefox 72.0.2 in Windows 10)); variations could be viewed as a better term than \"mutations\"; in the \"mutations\".\nA key aspect of the tool is that after applying the filters you can then either download the resulting set of PubMed IDs, or you can transfer (by literally copying and pasting) the IDs to view them in \"PubTator\", which opens in an iframe. I did find it variable whether \"PubTator\" would retrieve results or I would simply get a blank iframe. Also, some results give 1000s of PMIDs, which is clearly far too many for PubTator to cope with (again, blank iframe resulted). Several times I got results that said:\nWelcome! Guest. \| Log in\nResults: 1 to 3 of 3 1No Title\n\nPMID:{\"error\":\"API - Related citations ABSTRACT not availiable\n\n2No Title\n\nPMID:rate - Related citations ABSTRACT not availiable\n\n3No Title\n\nPMID:limit - Related citations ABSTRACT not availiable\n\n4No Title\n\nPMID:exceeded\",\"api-key\":\"130.14.18.113\",\"count\":\"4\",\"limit\":\"3\"} - Related citations ABSTRACT not availiable\n\nand that was simply copying and pasting 3 PMIDs (note typo in word \"available\").\nSo I am not convinced that the viewing in PubTator aspect, while potentially useful, is actually fully operational. Furthermore, when PubTator opens in the iframe it clearly says \"You will be automatically redirected to the new and improved PubTator Central (PubTator 2.0) website after January 2020. \" Well, it's definitely February and I wasn't redirected anywhere...\nThe methodology in general seems to rely heavily upon PubTator; the PubTator 2019 paper claims it is updated daily so it would be good to know how often CPP updates their initial \"PubTator\" data. Indeed, how often they update any of the data sources and how their update cycles run would be very helpful. I am also confused by Figure 1, as it has PubMed as a separate input from PubTator, but I understood that PubTator was based on PubMed? Surely concept-article associations have to have source articles in the first place?\nThe abstract states \"CPP currently includes information for ~1.1 million cancer-related publications associated with >23,000 human genes\" but then Table 1 and the text states 19,551 genes. Also, why 19,551 genes? This is never explained. The set appears to include pseudogenes and long ncRNAs so this could also be worth mentioning.\nI am also less than convinced about the usefulness of the \"cancer terms\" when they can be as general as \"patient\", \"DNA\", \"diagnosis\" etc, but I guess it is up to the user to assess their utility themselves. A lot of emphasis is placed on their inclusion in this tool, but in reality I am dubious about how useful they would actually be.\nI think there are far too many figures included, and mostly screenshots - these need to be condensed to show key information, or removed altogether.\nIn summary, I think this paper describes a tool that is a good concept, but the execution currently needs some more development and there are clearly some bugs. If these bugs were fixed and perhaps some UX testing done to improve the tool and the manuscript discussed this and dealt with the issues I have raised above, both would be greatly improved.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "5733", "date": "20 Jul 2020", "name": "Garrett M. Dancik", "role": "Author Response", "response": "I want to thank the reviewer for many constructive comments and for thoroughly testing the CPP tool. While we will not be able to formally revise and resubmit the manuscript at this time, we have made some minor changes to the tool and address some of the reviewer’s comments below: Comment: I used the URL listed for accessing the tool - in fact this URL then directs you to another URL, http://bioinformatics.eaternct.edu/app/CPP/. I have no idea why that URL isn't actually listed in the manuscript, and it was disappointing to see that they haven't bothered to make this https instead of http. Response: The URL provided in the manuscript, https://gdancik.github.io/bioinformatics/CPP/, is a stable URL and the homepage for CPP. We intentionally chose not to link directly to the tool in our manuscript, because computational demands may require moving the tool itself to a different host. The homepage will always link to the current tool. We also agree that https should have been used instead of http, and we have migrated our tool to https. Comment: A few comments on the user interface: the home button doesn't seem to take you anywhere - if you've run a search and click home you stay exactly where you are on the results page. Also clicking on \"Cancer Publication Portal\" doesn't take you anywhere; personally I find it quite irritating that the initial stages of any search, selecting your filters, involve a modal popup window, which then closes, it also makes modifying the terms clunky; Response: We have made a few changes to the interface that we hope improves the user experience. In place of a “Home” tab we now have a “Search” tab, which eliminates the initial modal popup. Following a search, the results are shown on the “Results” page. The user can go back to the “Search” page at anytime to carry out another search. We have also updated the “Cancer Publication” Portal link so that clicking on it reloads the page. Comment: the order of the genes listed in the drop-down seems very odd (I think it could be based on the NCBI Gene ID for each gene?) - it would make more sense to make the list alphabetical; Response: We appreciate this comment. The genes are now sorted alphabetically rather than by NCBI Gene ID. Comment: When selecting the \"terms\", it would be helpful to have an \"X\" next to each selected term to make it clear how they can be removed (clicking delete did remove the term but it also did something odd to the \"selected\" box, where a dropdown suddenly appeared...(using Firefox 72.0.2 in Windows 10)); Response: We have clarified in the instructions that terms may be removed by clicking on the table or by removing them from the dropdown box. Comment: A key aspect of the tool is that after applying the filters you can then either download the resulting set of PubMed IDs, or you can transfer (by literally copying and pasting) the IDs to view them in \"PubTator\", which opens in an iframe. I did find it variable whether \"PubTator\" would retrieve results or I would simply get a blank iframe. Also, some results give 1000s of PMIDs, which is clearly far too many for PubTator to cope with (again, blank iframe resulted). Response: We agree that viewing selected articles in PubTator is a key aspect of our tool, and our goal is for this process to be as seamless as possible. We have encountered similar API errors in the past, both while using CPP as well as when using PubTator directly. These errors are beyond our control. However, we now explicitly instruct users to go directly to PubTator (now PubTator Central) if encountering errors on our page. We also have tested our tool with PubTator Central and have been able to retrieve citations for >10,000 articles without any issues. Comment: The methodology in general seems to rely heavily upon PubTator; the PubTator 2019 paper claims it is updated daily so it would be good to know how often CPP updates their initial \"PubTator\" data. Indeed, how often they update any of the data sources and how their update cycles run would be very helpful. Response: Our original intention was to update our tool approximately once a month following the PubTator bulk data release schedule. This is mentioned at the end of our manuscript. However, PubTator has moved to PubTator 2.0 (PubTator Central), and while moving our pipeline to PubTator 2.0 (PubTator Central) was our intention, we are freezing our tool currently with the final release of PubTator (on 2/15/2020). While PubTator 2.0 offers improvements that are useful, its use of full text papers in our experience limits its usability for our purposes. In particular, text-mining (as of 7/3/2020) may identify terms that are mentioned outside of the main paper (e.g., the references or author contributions section). As a result, we have found the number of gene mentions to be inaccurate based on a testing set of genes. Comment: I am also confused by Figure 1, as it has PubMed as a separate input from PubTator, but I understood that PubTator was based on PubMed? Surely concept-article associations have to have source articles in the first place? Response: This should have been more clear in the figure and in the text. Our Figure 1 provides information about how data is integrated into CPP. The gene, mutation, chemical, and disease associations are downloaded from PubTator. While these associations are based on PubMed, PubMed is not our primary source for this data. However, the cancer term mentions are based on PubMed data which is our primary source. Comment: The abstract states \"CPP currently includes information for ~1.1 million cancer-related publications associated with >23,000 human genes\" but then Table 1 and the text states 19,551 genes. Also, why 19,551 genes? This is never explained. The set appears to include pseudogenes and long ncRNAs so this could also be worth mentioning. Response: The >23,000 number was a mistake on our part. The correct number (at the time of the initial publication), was 19,551 genes, which does include pseudogenes, long ncRNAs, and others. We include all genes (molecules assigned an ID by the HGNC) that are associated with at least one cancer publication based on PubTator." } ] }, { "id": "59613", "date": "21 Feb 2020", "name": "Qingyao Huang", "expertise": [ "Reviewer Expertise Protein-protein interaction prediction", "protein orthology", "text-mining", "statistical analysis of experimental data", "web/tool development." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe publication describes the web tool for search, retrieval and computing statistics of cancer publications related to the input gene(s). The tool fully builds upon open source data from PubTator which links medical and biological concepts to PubMed articles. The only derived data is the “Cancer Terms” associations data set. These are words (tags) that are significantly associated with the cancer related publications - that is - publications mentioning any cancer MeSH term (and at least one gene) significantly more relative to the all other publication (with at least one gene mentioned). Methodology is adequately described and the code is publicly available.\n\nInteraction with the website:\nThe user queries the database with a gene or a set of genes, and is presented with a list of cancer types associated with their list sorted by a number of associations. After the user selected the cancer type(s) of interest, it is presented with a bar graph with frequencies (counts) of these cancer types in the literature. Furthermore the user can filter the results by Cancer Terms, Drugs, Mutations and Additional Genes. Each of these filters allows the user to create a simple bar graph with the 10 most frequent term on one axis and number of articles on the other. The site is fast and responsive even for large gene list. All results can be downloaded and the code is publicly available.\nMain issues/limitation:\nThe main issue with the tool is lack of any statistical testing. The web tool only lists frequencies (counts) of these associations, which is not particularly informative. The tool should provide information if the user's initial gene or gene set is significantly more associated with particular cancer types/terms/drugs and how specific this association is, this results in an unspecific terms being always listed first. More crucially however, for example, a drug could have 10 associations with a user input gene out of a total of 10 association in the full corpus, or it could be 10 out of 10.000 in total. The interpretation of the results would substantially differ in both of these cases, however with the current state of the web tool, there is way to tell these two cases apart.\n\nAnother glaring limitation of the tool is that the only entry point is a gene or a gene set. There seem to be no good reason why the initial query would not be a cancer type, cancer term or the a drug. It’s would be a valid and potentially interesting question to ask the tool: Give me all the cancer types and genes associated with, for example, Roscovitine. As of now there is no way to generate such a data set as the output is limited to the associations with the entry gene-list.\n\nOther Issues/limitations:\nThe input gene selection box stops listing genes at the letter \"C\". Also the list is not properly sorted, there are rare other random genes beginning on T or S in between the sorted genes.\n\nWeb browsers back button doesn’t function properly (I guess it’s partially a limitation of the framework used, but the developers should avoid frameworks and tools that break basic browser UI functions - at very least the tool should warn the user that they work will be lost when the back button is pressed).\n\nThe user can’t share the state of the website (their results) with other users.\n\nWhen I click on “New Gene Search” I can’t select the same gene.\n\nWhen I double click on the cancer type in the “Select Cancer Types” window couple of times, I can force the app infinitely starts refreshing.\n\n“Cancer publication Portal” should be a hyperlink which sends the user to the start page.\n\niFrames, such as the ones used for PubTator should be avoided, except looking out of place and being confusing to the user, it’s an unstable solution as it may break functionality of framed websites such as pop-ups, full screen features (e.g. videos), and back button. In addition to that not all we browsers support iFrames, it’s regarded as unsafe, it breaks the webpage for the impaired users, and one cannot copy/paste the URL of the iframe which is a fundamental usability issue. Possible solution is to link directly to the search results. PubTator search result have simple GET requests scheme which could easily be implemented (the authors should be aware that there is an URL size limit and how many PMID PubTator can actually process, but these limitations also applies to the current solution).\n\nThe authors should consider text-mining NCIt Neoplasm Core instead relying in PubTator MeSH terms. The web tool focus is on oncology and cancer researchers. The Terminology provided by MeSH system is known for lacking granularity. NCIt has extensive hierarchy for cancer-related terms with high coverage. Utlilizing inferior ontology makes the tool less useful to the target audience.\n\nPubTator is already an outdated tool and is now developed further as Pubtator 2.0, which includes, among other improvements, more sophisticated text-mining and corpus of full text papers. The authors should move their pipeline to PubTator 2.0 system.\n\nIs the rationale for developing the new software tool clearly explained? No\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "5734", "date": "20 Jul 2020", "name": "Garrett M. Dancik", "role": "Author Response", "response": "I want to thank the reviewers for many constructive comments and for thoroughly testing the CPP tool. While we will not be able to formally revise and resubmit the manuscript at this time, we have made some minor changes to the tool and address some of the reviewer’s comments below: Comment: The main issue with the tool is lack of any statistical testing. The web tool only lists frequencies (counts) of these associations, which is not particularly informative. The tool should provide information if the user's initial gene or gene set is significantly more associated with particular cancer types/terms/drugs and how specific this association is, this results in an unspecific terms being always listed first. More crucially however, for example, a drug could have 10 associations with a user input gene out of a total of 10 association in the full corpus, or it could be 10 out of 10.000 in total. The interpretation of the results would substantially differ in both of these cases, however with the current state of the web tool, there is way to tell these two cases apart. Response: We appreciate this comment and have updated our tool to calculate and rank results by statistical significance. Specifically, an enrichment score is calculated for each term (cancer type, drug, etc), which measures how much more likely a term appears in the selected articles compared to all articles in the database. For example, a score of 4 means that the term is 4x as likely to appear in the title/abstract of selected articles than all cancer-related articles in the database. P-values, FDRs, and additional statistical information are provided for each set of results under the \"Full Table\" tab. Comment: Another glaring limitation of the tool is that the only entry point is a gene or a gene set. There seem to be no good reason why the initial query would not be a cancer type, cancer term or the a drug. It’s would be a valid and potentially interesting question to ask the tool: Give me all the cancer types and genes associated with, for example, Roscovitine. As of now there is no way to generate such a data set as the output is limited to the associations with the entry gene-list. Response: We acknowledge that this is a limitation of the tool, which was designed to be gene-centric in nature. The tool is appropriate for users wanting to summarize cancer-related articles containing one or more genes. Comment: The input gene selection box stops listing genes at the letter \"C\". Also the list is not properly sorted, there are rare other random genes beginning on T or S in between the sorted genes. Response: In our previous version of CPP, we had mistakenly sorted the genes by GeneID, rather than alphabetically by gene symbol. We have corrected this and now sort genes alphabetically. While the input box does not show all genes, the user can start typing into the text box to retrieve matching genes. This feature is now stated explicitly in the drop down label. Comment: Web browsers back button doesn’t function properly (I guess it’s partially a limitation of the framework used, but the developers should avoid frameworks and tools that break basic browser UI functions - at very least the tool should warn the user that they work will be lost when the back button is pressed). The user can’t share the state of the website (their results) with other users. Response: We acknowledge that these are current limitations of the tool, and are important features that may be incorporated in the future. We have added a note to the user on the welcome page that the Back button is not functional on the page. Comment: When I click on “New Gene Search” I can’t select the same gene. Response: This is intentional in order to reduce the computational burden on our server; if a user wants to “reset” a search, the user can clear the filters or click the “Cancer Publication Portal” link to refresh the page (see next item). Comment: “Cancer publication Portal” should be a hyperlink which sends the user to the start page. Response: Done Comment: iFrames, such as the ones used for PubTator should be avoided, except looking out of place and being confusing to the user, it’s an unstable solution as it may break functionality of framed websites such as pop-ups, full screen features (e.g. videos), and back button. In addition to that not all we browsers support iFrames, it’s regarded as unsafe, it breaks the webpage for the impaired users, and one cannot copy/paste the URL of the iframe which is a fundamental usability issue. Possible solution is to link directly to the search results. PubTator search result have simple GET requests scheme which could easily be implemented (the authors should be aware that there is an URL size limit and how many PMID PubTator can actually process, but these limitations also applies to the current solution). Response: We agree that iframes have limitations, but prefer them in this case since it makes viewing of search results easier. In addition, we provide a link to PubTator Central so that users can access the page directly if they prefer. Comment: PubTator is already an outdated tool and is now developed further as Pubtator 2.0, which includes, among other improvements, more sophisticated text-mining and corpus of full text papers. The authors should move their pipeline to PubTator 2.0 system. Response: Although moving our pipeline to PubTator 2.0 (PubTator Central) was our intention, as mentioned in the Discussion of our manuscript, we are freezing our tool currently with the final release of PubTator (on 2/15/2020). While PubTator 2.0 offers improvements that are useful, the use of full text papers in our experience limits its usability for our purposes. In particular, text-mining (as of 7/3/2020) may identify terms that are mentioned outside of the main paper (e.g., the references or author contributions section). As a result, we have found the number of gene mentions to be inaccurate, and in some cases by orders of magnitude, based on a testing set of genes." } ] } ]	1	https://f1000research.com/articles/8-2073
https://f1000research.com/articles/8-2065/v1	06 Dec 19	{ "type": "Research Article", "title": "Epidemiology of viral hepatitis B and C in Punjab, Pakistan: a multicenter cross-sectional study, 2017-18", "authors": [ "Amjad Khan", "Saira Afzal", "Aashifa Yaqoob", "Razia Fatima", "Mahboob Ul Haq", "Khunsa Junaid", "Abdul Nadir", "Saira Afzal", "Aashifa Yaqoob", "Razia Fatima", "Mahboob Ul Haq", "Khunsa Junaid", "Abdul Nadir" ], "abstract": "Background: Globally, approximately 240 million people are chronically infected with hepatitis B virus (HBV) and hepatitis C virus (HCV), which are responsible for 96% of all hepatitis-related mortality. Pakistan has the second highest prevalence of HCV in the world. Methods: We conducted this study to ascertain the prevalence and potential risk factors associated with HBV and HCV infections in Punjab. A multi-center cross-sectional study was conducted, involving 24 Hepatitis Prevention and Treatment Clinics of the Pakistan Kidney and Liver Institute and Research Center clinics, Lahore. A total of 141,705 individuals who visited the clinics during 2017-18 were included for seroprevalence analysis of hepatitis B (HBsAg) and C (Anti-HCV). In addition, 12,427 individuals from the main group underwent detailed face-to-face interviews based on a predesigned questionnaire for risk factor assessment. Results: The overall prevalence for HBV and HCV was 8.4% and 42.7%, respectively. Of those with HCV infection, 4.2% had a co-infection. The transgender population had a higher seroprevalence of HBV (11.8%) and HCV (58.8%). Higher HBV seroprevalence was found in a younger age group (16-30 years), while the older age group (>61 years) had a higher HCV seroprevalence. Geographically, Muzaffargarh district had the highest HBV seroprevalence at 26%, followed by Rajanpur district (20.3%). HCV seroprevalence was significantly (P value <0.05) higher in Shujabad district (66.4%), followed by Muzaffargarh (65.2%). Using multivariable logistic regression analysis, age, gender, intravenous injections, hijama therapy, dental procedure, circumcision by barbers, barber shaving, hospitalization and having had surgical procedures were all found to be significant risk factors (OR >1; p<0.05) for HBV and HCV. Conclusions: There is an urgent need for proper implementation of preventive and control strategies, as well as formal evaluation and monitoring mechanisms. Sustainable and adequate funding of public-sector hepatitis programs is also an extremely important area that should not be neglected.", "keywords": [ "Epidemiology", "viral hepatitis", "seroprevalence", "risk factors", "Pakistan" ], "content": "Abbreviations\n\nHCV, Hepatitis C Virus; HBV, Hepatitis B Virus; GHSS, Global Health Sector Strategy; IDU, Injectable drug usage; PKLI-RC, Pakistan Kidney and Liver Institute and Research Center; HPTP, Hepatitis Prevention and Treatment Program; HPTC, Hepatitis Prevention and Treatment Clinic; EMR, Electronic Medical Record\n\n\nIntroduction\n\nHepatitis B (HBV) and hepatitis C (HCV) viral infections are major global health problems. Globally, approximately 240 million people are chronically infected with HBV and 130–150 million with HCV1,2. Hepatitis B and C are responsible for 96% of all hepatitis-related mortality, leading to an estimated 1.45 million deaths annually (WHO, 2015). The consequences of chronic viral hepatitis and HIV infections are among the top ten lethal infections worldwide1. Globally, 80% of the HCV burden is concentrated in low and middle-income countries (LMICs)3. However, published data suggest that a large proportion of HBV4 and HCV positive patients are not aware of their serostatus in developing countries, especially those belonging to low income households5. Furthermore, community-based studies indicate low screening uptake and considerable numbers of dropouts in both HBV and HCV care continuums6. The Global Health Sector Strategy (GHSS) on viral hepatitis stresses the need for aggressive targets for eliminating viral hepatitis as a public health threat by 2030 and has proposed a target of a 90% reduction in incidence and 65% reduction in mortality due to chronic HBV and HCV infections7. The higher prevalence of HBV and HCV in developing countries is also attributable to fragile health and hepatology services2, in addition to multiple community-based risk factors.\n\nInjectable drug usage (IDU) and associated sharing of contaminated injecting equipment8, blood donation9, unscreened blood transfusion10, surgical procedures11, sharp object injuries12, tattooing13 and barbering14 have all been reported as risk factors for the occurrence and transmission of HBV and HCV infections. The cumulative risk of HCV infection in a population often increases with age as well15.\n\nPakistan has the second highest prevalence of HCV (5%) after Egypt and the second highest number of people suffering from HCV after China16. In 2007–08, a nation-wide survey reported a general population prevalence of 2.5% and 4.9% for HBV and HCV, respectively. At a provincial level, Punjab has the highest burden of hepatitis17. The current study was conducted to report on the prevalence and risk factors associated with HBV and HCV infections in Punjab. The findings of this study will help in further understanding of the local epidemiology of hepatitis and its prevention and control.\n\n\nMethods\n\nEthical approval was obtained from the institutional review board (IRB) of the Pakistan Kidney and Liver Institute and Research Center (PKLI-RC; approval # PKLI-73). A copy of the consent form is provided as Extended data. Written consent was obtained from all the participants prior to interviews for the use of their epidemiological data for research and publication. Parental consent from minor participants was obtained for participants aged 16 years or below. It was signed by the parent or guardian who accompanied the patient on behalf of the minor. This consent form was provided in both the local language and in English. A copy of the minor consent form duly signed by the principle investigator of study was also provided to the parents of the minor participant. In cases where parents were not willing to participate in the study and did not sign the minor consent form, the patients were not included in the study.\n\nPunjab is one of the most populated provinces of Pakistan, with a population of 110 million18, and has a high burden of hepatitis B and C. The PKLI-RC is state-of-the-art tertiary care center in Punjab’s provincial capital, Lahore. The hospital is currently providing clinical services for liver and kidney diseases, including kidney transplants. Given the burden of hepatitis B and C in the province, the PKLI-RC has established an outreach program (Hepatitis Prevention and Treatment Program), which has been providing free-of-charge, evidence-based hepatitis B and C preventive, screening, diagnostic, vaccination and treatment services to the population of Punjab since March 2017. The Hepatitis Prevention and Treatment Program’s (HPTP) services are being delivered through 24 Hepatitis Prevention and Treatment Clinics (HPTCs), which are based in Lahore and 23 other districts of the Punjab province. In addition to the PKLI-RC’s HPTP, two other similar government-funded hepatitis control programs are providing necessary care services to the population of Punjab. This cross-sectional study is based on the data of individuals who visited the HPTCs from March 2017 to August 2018 for screening, diagnosis and treatment of hepatitis.\n\nAll the visiting individuals were screened for HBV and HCV after their registration in the HPTCs’ Electronic Medical Record (EMR). Patients were included in the study after signing the consent form once the study had been explained to them by the investigator19. Patients who did not sign the consent form to participate in the study were screened and treated but they were not included in the study analysis. Patients were included irrespective of their age, gender, ethnicity and locality within Punjab Province. Patients were excluded from the study if they were unwilling to participate in the study or came from any other province of the country. Face-to-face interviews were conducted after voluntary informed consent was given. A total of 141,705 individuals were registered and screened in at all HPTCs and were then interviewed at the health center, the majority being carried out at the HPTC-Lahore clinic. After signing the consent form, patients were interviewed about their exposures to risk factors by trained interviewers and/or medical officers using a questionnaire20. Each question was made clear to the patient in order to get the true answer according to the patient’s knowledge and memory. The criteria for inclusion in further analysis was no previous history of hepatitis. Of the 141,705 patients interviewed, only 12,427 met the criteria and only their data was used in the final analysis.\n\nThe predictor variables included in the analyses were: age (in years); gender (male/female/transgender); ever having had intravenous (IV) injections (and number of injections in the last five years), ever having had blood transfusions, ever having had dental procedures, being hospitalized within the last five years, ever having had surgery, having taken recreational drugs in the last five years, having had a circumcision performed by a barber (males only), shaving by barbers, visiting a beauty parlor within the last five years, ever having had hijama therapy, having a body piercing in the last five years, having a tattoo in the last five years and ever having performed self-flagellation with sharp objects. The outcome variables were serostatus for hepatitis B (HBsAg) and hepatitis C (anti-HCV).\n\nThe data was extracted from a centralized electronic medical records (EMR) database in Excel format. An epidemiological questionnaire, also part of the EMR system, was used to collect the data on individuals’ demography and hepatitis-related risk factors. The questionnaire was piloted on 30 participants to obtain estimates about the expected response rates, data quality, validity and comprehensibility of the questionnaire. These participants were patients who visited these clinics routinely and were coming for a screening or checkup. They were provided with a consent form for their willingness to participate in the study. After signing the consent form, they were interviewed by medical officers using the pilot questionnaire. After reviewing the results of piloted questionnaire, a few variables were excluded from the final questionnaire (are you taking drugs; have you been bitten by any animal in last five years; are you taking cannabis) because these variables were found to be irrelevant and response rate for these variables were very low. The overall response rate was 96.66% (29/30) and the data quality was good in terms of dimensions such as ‘consistency’, ‘accuracy’, ‘completeness’, and ‘timeliness’. The internal consistency was measured by Cronbach’s alpha, with an alpha value of 0.87. The normality of data was assessed for the pilot survey to evaluate the fitness of data.\n\nQuestionnaires were completed by staff during face-to-face interviews, conducted in the local language by a trained interviewer. Interviewers were bachelor nurses or medical officers who had been trained by an epidemiologist to carry out interviews. The data was exported to Epi-Data for revisions such as data cleaning and data validation. Where >10% of all variables in any patient’s questionnaire were missing or not provided, the patient was excluded from analysis.\n\nA 5ml blood sample was collected from each patient using disposable vacuum syringes by nurses at the health centers. Samples collected at the sentinel sites were transported daily to the central laboratory for laboratory diagnosis. Serum specimens from all samples were separated using standard protocols and stored at 20°C on the same day until tested for HBsAg or Anti-HCV in the central PKLI laboratory in Lahore following WHO standard procedures. HBsAg and anti-HCV analyses were performed, the details of which are given below.\n\nA chemiluminescent micro particle immunoassay (CMIA) anti-HCV assay (ARCHITECT Anti-HCV assay, ABBOTT Diagnostics; Catalog no. 6L47) was conducted for the qualitative detection of antibodies against HCV in the patients’ serum and plasma. Recombinant HCV sample antigen-coated paramagnetic micro-particles and anti-human acridinium-labeled conjugates are fixed to create the reaction mixture and then incubated for 5 minutes at 25°C. Ancillary wash buffer was added to the mixture after washing, followed by another wash cycle and incubation for 5 minutes at 25°C. Pre-Trigger (Pre-Trigger Solution containing 1.32% w/v hydrogen peroxide) and Trigger solutions (containing 0.35N sodium hydroxide) are added to the mixture. The resulting chemiluminescent reaction was measured in relative-light-units (RLUs) using a luminometer. The presence of anti-HCV antibodies is determined by comparing the signals shown in the reaction to an active-calibration curve. If the signal in the specimen is greater or equal to the cutoff signal, the sample is then considered anti-HCV positive and vice versa. The ARCHITECT Anti-HCV assay calculates a result based on Sample RLU/Cutoff RLU (S/CO). The cutoff calculation is done such that; calibrator 1 Mean RLU Value × 0.074 = Cutoff RLU. Specimens with S/CO values <1.00 are considered nonreactive by the ARCHITECT Anti-HCV assay and need not be tested further. Specimens with S/CO values ≥ 1.00 are considered reactive by the ARCHITECT Anti-HCV assay. A two-step immunoassay for qualitative detection of HBsAg using CMIA technology was used. The ARCHITECT HBsAg assay (Catalog no. 6C36) utilized a four parameter logistic curve-fit (y-weighted) to generate a calibration curve. Specimens with concentration values < 0.05 IU/mL was considered nonreactive by the criteria of ARCHITECT HBsAg. Specimens with concentration values ≥ 0.05 IU/mL were considered reactive by the criteria of ARCHITECT HBsAg.\n\nData was analyzed using SPSS version 22 (IBM, NY, USA). Results are reported as percentages and odds ratios. Descriptive statistics of prevalence are reported in Table 1. To analyze the association of independent variables with the outcome variables, logistic regression was performed21. Variables with a significance level of <0.25 were retained in the final logistic model. Regression analysis of the data was conducted using multivariable logistic regression model. Goodness of fit of the model used was assessed using the Hosmer-Lemeshow test and the likelihood-ratio test. QGIS Version 3.2.222 was used for geographical distribution and heat maps were developed based on coordinates data collected during interviews to present the seroprevalence of HBV and HCV at the district level.\n\nHBV, hepatitis B virus; HCV, hepatitis C virus; n, number of participants.\n\n\nResults\n\nA total of 141,705 individuals were included in the analysis, including 64,622 (45.6%) male, 77,066 (54.3%) female and 17 transgender (0.01%) patients23. All the patients were screened for both HBV and HCV. The study population was distributed into five age groups, with 31–45 making up around 34% of the total, as highlighted in Table 1. Of these, data from 12,427 participants were included in further analysis24. Overall 11,157 (89.7%) participants self-reported having had IV injections, with around 31% reported having more than 10 IV injections.\n\nDescriptive statistics were Figure 1 gives the overall prevalence of HBsAg and anti-HCV in the study population, which was 8.4% and 42.7%, respectively. Of those with HCV infection, seroprevalence of co-infection with HBV was recorded to be 4.2%. The seroprevalence data stratified by various categories is presented in Table 1. Briefly, the prevalence of co-infection was higher in males (5.7%) than females (3.2%). The transgender population had a significantly higher seroprevalence of HBsAg (11.8%) and anti-HCV (58.8%) compared to other gender groups. The chi-square test found a directly associated trend of increased HCV and co-infection was found with increasing age. Geographical heat maps for the seroprevalence of HBV and HCV are presented in Figure 2. Muzaffargarh district had the highest HBV seroprevalence at 26%, followed by Rajanpur (20.3%), Lodhran (10.6%), and Shujabad (10.3%). HCV seroprevalence was significantly higher (P value <0.05) in Shujabad district (66.4%), followed by Muzaffargarh (65.2%), Nankana Sahab (62.5%), and Lodhran (61.8%).\n\nRight: heat maps of HBV (A) and HCV (B) seroprevalence in population visiting HPTP clinics in 2017/18, Punjab Province, Pakistan (maps are based on the data collected in this study during interviews).\n\nIn the multivariable logistic model, age group (16–30 years) was found to be a significant (p-value <0.05) risk factor (OR=4.2) for HBV occurrence, followed by age groups 31–45 years (OR=3.4), and >61 group (OR=3.2). Males had 2.1-fold higher risk of HBV infection than females. The other risk factors found to be significantly associated with the occurrence of HBV included exposure to hijama therapy, circumcision performed by barbers, barber shaving, recreational drug use, tattooing, beauty parlor visits, IV injections and having >10 injections (Table 2).\n\nOR, odds ratio; C, 95% confidence interval; HBV, hepatitis B virus; IV, intravenous.\n\nHCV occurrence was significantly associated with an increase in age of the patients. Belonging to the older age group (>61) was found to be a risk factor (OR=56.5; 95% C. I=40.6-78.6) for HCV infection. Exposure to hijama therapy was also recorded as a risk factor (OR=6.0). Females had a 1.3-fold higher risk of HCV than males. The other significant risk factors for HCV included circumcision by barbers, recreational drug usage, tattooing, piercing, blood transfusion, dental procedures, surgical procedures and prior hospitalization (Table 3).\n\nOR, odds ratio; CI, 95% confidence interval; HCV, hepatitis C virus; IV, intravenous.\n\nAge showed a significant association with HBV and HCV co-infection, as before. Patients in the >61 years age group had a 29.3-fold (95% C. I=6.9-124.6) higher risk of co-infection than the younger age groups. The other significant risk factors included barber shaving, self-flagellation, recreational drug use, circumcision by barbers, piercing, beauty parlor visits, history of blood transfusion and having >10 IV injections (Table 4).\n\nOR, odds ratio; CI, 95% confidence interval; HBV, hepatitis B virus; HCV, hepatitis C virus; IV, intravenous.\n\n\nDiscussion\n\nViral hepatitis is an important global public health problem. Several community-based studies report a higher prevalence of viral hepatitis in Pakistan both in Sindh and Punjab Province13–14,25. Studies on understanding the epidemiology of viral hepatitis and related risk factors in Pakistan are limited. The present study is an attempt to assess the epidemiology and the potential risk factors in individuals who visited PKLI-RC’s clinics during 2017–18.\n\nWe have found a very high prevalence in our clinic cohorts compared to the previous study reports in Pakistan25,26. Several social factors are responsible for the higher prevalence of HBV and HCV, including lack of health and safety standards due to unsatisfactory awareness and knowledge of the disease in the general population, as reported by 27. The poor literacy rate of 43% in Pakistan25 is also a reason for a higher prevalence of viral hepatitis. Areas with low literacy rates were found to have a higher prevalence (P value <0.05), as the awareness level of the population in these areas is low and these areas are exposed to the driving risk factors at a higher rate as compared to developed areas25,28. The higher prevalence of HBV and HCV in the transgender population could be due to a lower socioeconomic status and behavioral factors including alcoholism, promiscuity and IV drugs usage25,28. These behavioral factors are practiced more in transgender community and among the groups who are in contact with transgender community at regular basis7. The increasing prevalence of HBV and HCV in the transgender community is a concerning situation for an otherwise healthy population. Furthermore, the higher prevalence of hepatitis is also linked to the improper disposal of hospital waste in Pakistan29. This matter is now being aggressively tackled with the approval and implementation of the Hepatitis Act in Punjab.\n\nWe have found age to be a significant risk factor associated with the occurrence of HBV (Table 2). The younger age group (16–30 years) was at a 4.2-fold higher risk of HBV than the older age groups. The variation in the seroprevalence of HBV in different age groups could be due to the changing immune response of the body to infectious agents at certain ages, as also supported by the findings reported previously (Cheng et al., 2007). We also found males to be at a higher risk and barber shaving was significantly associated with HBV occurrence. Exposure to barber shaving, IV injections, and hijama therapy could be promoting the risk of HBV occurrence in males in our study population. Frequent exposure to barber shaving, surgical and dental procedures, blood donation and a higher number of injections being important risk factors for HCV infection has been reported elsewhere10. Our results are in line with the findings reported from other regions of the world21,25,30.\n\nHepatitis C viral infection has been reported to be an emerging epidemic in Pakistan25. Here, an elevating trend of HCV seroprevalence was recorded with an increase in age (Table 3). The highest seroprevalence of HCV was recorded in age group ≥61 years, followed by age group 46–60. This is likely due to a cumulative effect whereby the exposure to multiple risk factors increases with age. For instance, the number of child births, recreational drug use, exposure to barbers and numbers of injections all increase with age. These results are in line with the findings of 29. Hijama therapy was identified as a potential risk factor for HCV infection. This might be due to unhygienic environments where the procedure is carried out and the use of unsterilized instruments that are not properly autoclaved and are used on multiple individuals27. Circumcision performed by barbers was also recorded as a risk factor in our study. This could be attributable to the use of contaminated instruments on multiple individuals26. Other exposure variables found in this study have previously been reported, such as history of hospitalization, surgical procedures, recreational drug use, IV injections, tattooing, piercing, barbers shaving, beauty parlor visit, blood transfusion and self-flagellation with sharp objects31,32.\n\nThe main strength of this study is that this involved 24 clinics based in various parts of the province and had a large sample size of 141,705 individuals, who were screened for HBV and HCV infections through ELISA. Data was collected through a centralized EMR system assuring the accuracy, completeness, consistency, reliability and repeatability of the data. Of the 141,705-study population, we only interviewed 12,427 participants for identification of risk factors associated with viral hepatitis. However, this sizeable number is still large enough to provide us reliable estimates (Table 2–Table 4) on the role of various risk factors in the occurrence and spread of hepatitis in the local population. The limitations of the study are that all 141,705 individuals were not interviewed due to loss to follow up or low response levels in signing the consent form.\n\n\nConclusions\n\nThere is an urgent need for the implementation of preventive and control strategies to halt the rapid increase in seroprevalence of HBV and HCV in Punjab. A considerable amount of work is already being done to tackle the issue including, but not limited to, screening of donated blood, proper sterilization of health care instruments, implementation of hospital waste management systems, training of barbers, improving the uptake of birth dose of hepatitis B vaccination, and the implementation of the Punjab Hepatitis Act. These initiatives also need to be supported by improvements in operational research, such as in evaluating and monitoring their impact in decreasing the spread of viral hepatitis. Health professionals must be trained to disseminate accurate information about viral hepatitis. Sustainable and adequate funding of the public-sector hepatitis programs is also an extremely important area that should not be neglected.\n\n\nData availability\n\nFigshare: Epidemiology of viral hepatitis B and C in Punjab, Pakistan: a multi-center cross-sectional study, 2017–18 (Extended Data File). https://doi.org/10.6084/m9.figshare.9891260.v123\n\nThis project contains the following underlying data:\n\n- Extended Data File F1000.xlsx (raw data for all 141,705 individuals interviewed)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: Epidemiology of Viral Hepatitis B and C in Punjab, Pakistan: A Multicenter Cross-sectional Study, 2017–18. https://doi.org/10.6084/m9.figshare.9702845.v124\n\nThis project contains the following underlying data:\n\n- Data file for F1000.xlsx (raw data for the 12,427 individuals included in final analysis)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nFigshare: Questionnaire.docx for Epidemiology of viral hepatitis B and C in Punjab, Pakistan: a multi center cross-sectional study, 2017–18. https://doi.org/10.6084/m9.figshare.9993044.v120\n\nFigshare: Epidemiology of viral hepatitis B and C in Punjab, Pakistan: a multicenter cross-sectional study, 2017–18 (Consent Form). https://doi.org/10.6084/m9.figshare.9730592.v219\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe are thankful to the data collectors and IT department of PKLI-RC for their support during data extraction and validation.\n\n\nReferences\n\nGower E, Estes C, Blach S, et al.: Global epidemiology and genotype distribution of the hepatitis C virus infection. J Hepat. 2014; 61(1 Suppl 1): S45–S57. PubMed Abstract \| Publisher Full Text\n\nLim AG, Qureshi H, Mahmood H, et al.: Curbing the hepatitis C virus epidemic in Pakistan: the impact of scaling up treatment and prevention for achieving elimination. Int J epidemiol. 2018; 47(2): 550–560. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO: Global Health Sector Strategy on Viral Hepatitis 2016-2021. 2017. Reference Source\n\nChang TT, Lai CL, Kew YS, et al.: Entecavir treatment for up to 5 years in patients with hepatitis B e antigen-positive chronic hepatitis B. Hepatology. 2010; 51(2): 422–30. PubMed Abstract \| Publisher Full Text\n\nAllard NL, MacLachlan JH, Cowie BC: The cascade of care for Australians living with chronic hepatitis B: measuring access to diagnosis, management and treatment. Aust N Z J Public Health. 2015; 39(3): 255–9. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Guidelines for the prevention, care and treatment of persons with chronic hepatitis b infection. Geneva: World Health Organization. 2015. Reference Source\n\nWorld Health Organization: Guidelines for the screening, care and treatment of persons with hepatitis C infection. Geneva: World Health Organization. 2014. PubMed Abstract\n\nMathers BM, Degenhardt L, Ali H, et al.: HIV prevention, treatment, and care services for people who inject drugs: a systematic review of global, regional, and national coverage. Lancet. 2010; 375(9719): 1014–28. PubMed Abstract \| Publisher Full Text\n\nLiaw YF, Gane E, Leung N, et al.: 2-year GLOBE trial results: telbivudine Is superior to lamivudine in patients with chronic hepatitis B. Gastroenterology. 2009; 136(2): 486–95. PubMed Abstract \| Publisher Full Text\n\nHawk L, Norton BL, Cunningham CO, et al.: The Hepatitis C virus treatment cascade at an urban postincarceration transitions clinic. J Viral Hepat. 2016; 23(6): 473–478. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNelson PK, Mathers BM, Cowie B, et al.: Global epidemiology of hepatitis B and hepatitis C in people who inject drugs: results of systematic reviews. Lancet. 2011; 378(9791): 571–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen CJ, Iloeje UH, Yang HI: Long-term outcomes in hepatitis B: the REVEAL-HBV study. Clin Liver Dis. 2007; 11(4): 797–816, viii. PubMed Abstract \| Publisher Full Text\n\nJafari S, Copes R, Baharlou S, et al.: Tattooing and the risk of transmission of hepatitis C: a systematic review and meta-analysis. Int J Infect Dis. 2010; 14(11): e928–40. PubMed Abstract \| Publisher Full Text\n\nAli M, Idrees M, Ali L, et al.: Hepatitis B virus in Pakistan: a systematic review of prevalence, risk factors, awareness status and genotypes. Virol J. 2011; 8: 102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKiani RA, Anwar M, Waheed U, et al.: Epidemiology of Transfusion Transmitted Infection among Patients with β-Thalassaemia Major in Pakistan. J Blood Transfus. 2016; 2016: 8135649. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSievert W, Altraif I, Razavi HA, et al.: A systematic review of hepatitis C virus epidemiology in Asia, Australia and Egypt. Liver Int. 2011; 31 Suppl 2: 61–80. PubMed Abstract \| Publisher Full Text\n\nQureshi H, Mohamud BK, Alam SE, et al.: Treatment of hepatitis B and C through national programme--an audit. J Pak Med Assoc. 2013; 63(2): 220–4. PubMed Abstract\n\nCensus 2017: Human Population Census. Islamic Re-Public of Pakistan. 2017.\n\nKhan A, Afzal S, Yaqoob A, et al.: Epidemiology of viral hepatitis B and C in Punjab, Pakistan: a multicenter cross-sectional study, 2017-18 (Consent Form). 2019. http://www.doi.org/10.6084/m9.figshare.9730592.v2\n\nKhan A: Questionnaire.docx for Epidemiology of viral hepatitis B and C in Punjab, Pakistan: a multi center cross-sectional study, 2017-18. 2019. http://www.doi.org/10.6084/m9.figshare.9993044.v1\n\nKhan AJ, Luby SP, Fikree F, et al.: Unsafe injections and the transmission of hepatitis B and C in a periurban community in Pakistan. Bull World Health Organ. 2000; 78(8): 956–63. PubMed Abstract \| Free Full Text\n\nQGIS development Team: QGIS Geographic Information System. Open Source Geospatial Foundation. 2009.\n\nKhan A, Afzal S, Yaqoob A, et al.: Epidemiology of viral hepatitis B and C in Punjab, Pakistan: a multi-center cross-sectional study, 2017-18 (Extended Data File). 2019. http://www.doi.org/10.6084/m9.figshare.9891260.v1\n\nKhan A, Afzal S, Yaqoob A, et al.: Epidemiology of Viral Hepatitis B and C in Punjab, Pakistan: A Multicenter Cross-sectional Study, 2017-18. 2019. http://www.doi.org/10.6084/m9.figshare.9702845.v1\n\nAkhtar H, Badshah Y, Akhtar S, et al.: Prevalence of hepatitis B and hepatitis C Virus infections among male to female (MFT) transgenders in Rawalpindi (Pakistan). Adv Life Sci. 2018; 5(2): 46–55. Reference Source\n\nUllah Q, Khan K, Saeed K, et al.: Prevalence of Hepatitis C and B in MURCY Hospital Peshawar , KP , Pakistan. J Entomology and Zoo Studies. 2017; 1081–4. Reference Source\n\nLuby SP, Qamruddin K, Shah AA, et al.: The relationship between therapeutic injections and high prevalence of hepatitis C infection in Hafizabad, Pakistan. Epidemiol Infect. 1997; 119(3): 349–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMangla N, Mamun R, Weisberg IS: Viral hepatitis screening in transgender patients undergoing gender identity hormonal therapy. Eur J Gastroenterol Hepatol. 2017; 29(11): 1215–1218. PubMed Abstract \| Publisher Full Text\n\nRiaz H, Riaz T, Ullah F, et al.: Assessment of the seroprevalence of viral hepatitis B, viral hepatitis C and HIV in multitransfused thalassaemia major patients in Karachi, Pakistan. Trop Doct. 2011; 41(1) 23–25. PubMed Abstract \| Publisher Full Text\n\nAli SA, Donahue RM, Qureshi H, et al.: Hepatitis B and hepatitis C in Pakistan: prevalence and risk factors. Int J of Infect dis. 2009; 13(1): 9–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrown CR, MacLachlan JH, Cowie BC: Addressing the increasing global burden of viral hepatitis. Hepatobiliary Surg Nutr. 2017; 6(4): 274–276. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSharew NT, Mulu GB, Habtewold TD, et al.: Occupational exposure to sharps injury among healthcare providers in Ethiopia regional hospitals. Ann Occup Environ Med. 2017; 29(1): 7. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "61796", "date": "14 Apr 2020", "name": "Arshad Altaf", "expertise": [ "Reviewer Expertise Both experimental and non experimental research. However", "the statistical section certainly needs to be reviewed by a qualified person." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract\nBackground\nPlease use HBV and HCV estimates from the WHO fact sheets which are more updated.\n\nResults\n\nFor clarity it is suggested to expand what the authors mean by \"intravenous injections\" as many readers might associate it with intravenous drug use. If it is intravenous therapeutic or medical injections please state that.\n\nConclusions\nPlease make the conclusion more objective. When authors say \"monitoring mechanism\" please clarify monitoring of what? Public sector funding for what?\n\nIntroductions\n\nPlease use the HBV and HCV estimates from these WHO factsheets.\n\nHBV HCV\n\nThe most common terms nowadays is people who inject drugs (PWIDs) or injection drug user (IDU) however, PWID has replaced IDU.\n\nData variables\nPlease clarify if the question only asked intravenous injections and information regarding receiving intra muscular injections was not collected? In order to be clear it is suggested to rephrase ever having had intravenous (IV) injections (and number of injections in the last five years) to number of intravenous injections received in the last five years. Rephrase: ever having had blood transfusions to ever had blood transfusion. Applicable to all similar variables.\n\nData collection and management\n\nGenerally, so much detail about piloting is not required. In the interest of “economy of words” it is suggested to simply say, “the questionnaire was piloted and participants of the pilot were not included in the actual study.”\n\nSerum sample collection and diagnosis\n\nI am not qualified to review this section. However, if it can be reduced to fewer lines by the authors it will be beneficial for the reader. But if the authors are convinced that it should stay, I am okay with that.\n\nResults\n\nWhat do the authors mean by descriptive prevalence?\n\nThe standard way of presenting results is by describing demographic characteristics of the subjects which can also be developed by hepatitis status wise.\n\nTables should provide more information about the participants.\n\nNot clear what does a \"heat map\" mean? It seems like a distribution map of hepatitis status of the patents district wise. The map has been developed based on hepatitis status and district of origin of the patients.\n\nHow GPS coordinates were collected when all interviews were conducted at the health facility?\n\nThe flow chart provides the process of study i.e. the number of patients came in the health centres during the study period, number refused, number screened then perhaps the number interviewed. At the moment the flow chart and the tables seem the same. They have to be different.\n\nIn \"seroprevalence of HBV, HCV and co infection\" there is incorrect use of Chisquare. Chisquare shows association between two variables not trends.\n\nTables 2 to 4 showing crude OR as adjusted OR? Confidence intervals have 1 in the range which is insignificant but it is shown as significant.\n\nDiscussion\n\nDiscussion should be in this pattern:\nFirst para describing the key findings of the study. Subsequent paras talk about key findings in light of other peer reviewed studies. Penultimate para should be the limitation in which also authors have to mention \"recall bias\".\n\nIn conclusion please mention the use of availability of direct acting antivirals (DAAs) in Pakistan particularly for hepatitis C.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "64814", "date": "29 Jun 2020", "name": "Mohammad Farahmand", "expertise": [ "Reviewer Expertise Medical virology and epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript entitled: \"Epidemiology of viral hepatitis B and C in Punjab, Pakistan: a multicenter cross-sectional study, 2017-18\" was aimed to assess the prevalence of HBV and HCV infections in individuals who visited the Hepatitis Prevention and Treatment Clinics (HPTCs) in Punjab, Pakistan. The authors also performed a multivariate analysis to analyze potential risk factors. The study was conducted on a large sample size of 141,705 individuals. After reviewing the above manuscript in detail, I consider the manuscript well-written, and the arguments are developed in an orderly manner. Still, there are some concerns about this paper to be addressed.\nIn the \"Statistical analysis\" section, the authors claimed that variables with a significance level of <0.25 were maintained in the final logistic model, but all variables are included in tables 2,3, and 4. Please explain whether all variables in all tables have a significance level of <0.25.\n\nIn addition to multivariate analysis, please include the univariate logistic regression model results in tables 2,3, and 4.\n\nIn the result section, part: \"Seroprevalence of HBV, HCV and co-infection\" line 1, it seems figure 1 was not appropriately cited in the text.\n\nIn the discussion section, the authors try to compare their results that using samples from hospitals with hepatitis disorders (current manuscript), with the data from Akhtar et al. (ref 25) that estimates the prevalence of HCV and HBV infection in transgender males. Any comparison between these two groups, because of using different sampling population is not valid.\n\nIn the discussion section, part: \"Epidemiology of hepatitis risk factors\": for probable reasons for elevating trend of HCV seroprevalence with an increase in age, the authors should consider that about 70% of people infected with HCV will develop chronic HCV infection. So, they may remain seropositive for several years.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-2065
https://f1000research.com/articles/8-2062/v1	05 Dec 19	{ "type": "Research Article", "title": "Computational study of the interactions among structural analogues of acyl homoserine lactones (AHLs) and the Agrobacterium tumefaciens TraR binding site", "authors": [ "Daniela Pérez", "Maicol Ahumedo", "Eileen Herrera", "Catalina Vivas-Gomez", "Ricardo Vivas-Reyes", "Daniela Pérez", "Maicol Ahumedo", "Eileen Herrera", "Catalina Vivas-Gomez" ], "abstract": "Background: In the present investigation, relationships between a set of 34 analogues of N-acyl-L-homoserine lactones (AHL) and the TraR receptor were studied. The aim was to use molecular modeling as a strategy for elucidating important aspects of the mechanism of chemical signaling in the Gram-negative bacteria Agrobacterium tumefaciens, with the idea of identifying some of analogues’ structural characteristics and molecular interactions with the active site of the TraR receptor. Methods: For this purpose, we combine two molecular modeling strategies: molecular docking and three-dimensional quantitative structure-activity relationship (3D-QSAR). First, the molecular docking methodology was applied to a series of 34 analogues of AHL on the TraR transcriptional receptor to simulate the binding of analogues at the active TraR site. Secondly, 3D-QSAR models were generated to describe the correlation with the experimental biological activity using partial least squares (PLS) calculations and steric and electrostatic properties, which theoretically predict the activity of the 34 AHL analogues through statistical parameters and evaluate the prediction of the models obtained. Two alignment models were constructed; one using the optimized structures of the 34 analogues (ligand-based model) and another using the conformations of the best poses generated in the docking with TraR (receptor-based model). Results: The outcomes obtained for each protein-ligand complex showed that the Aspartic acid 70 and Threonine 129 residues are residues that participate in the formation of hydrogen bonds, while residues Alanine 38, Leucine, 40, Tyrosine 53, Glutamine 58, Tyrosine 61, Phenylalanine 62 and Valine 72 form hydrophobic interactions. These interactions are important in determining the antagonistic activity of the analogues under study against TraR. Conclusions: The ligand-based model produces better statistical results expressed in terms of several rigorous evaluation criteria, such as Q2 and R2 for the data sets than those of the receptor-based model.", "keywords": [ "Active TraR site", "Quorum Sensing", "Molecular Docking", "3D-QSAR" ], "content": "Introduction\n\nThe intracellular communication mechanism known as quorum sensing (QS) makes it easier for bacteria to develop cooperative behaviors, which coordinate their activities in order to function as a multicellular unit1. QS is a process based on the synthesis, diffusion, and perception of small signal molecules called autoinducers (AIs), through which a bacterial population can monitor their cell density and regulate specific gene expression in response to local changes in its population density2,3. Bacterial behaviors regulated by QS include the secretion of virulence factors, biofilm formation, bioluminescence, sporulation and conjugation4.\n\nQS in Gram-negative bacteria is mediated by N-acyl homoserine lactones (AHLs). In this AHL-dependent system, the QS signal is detected by a cytosolic transcription factor (R). AHL synthase (Protein I) produces AHL molecules that can diffuse through the cell membrane and as they reach a critical extracellular concentration, they enter the cell again and bind to the R protein, forming the R-AHL complex, which activates the expression of some specific genes5,6.\n\nThe Gram-negative bacterial species Agrobacterium tumefaciens regulates the expression of the genes necessary for the replication and conjugation of the tumor-inducing plasmid in dicotyledonous Ti plants through the QS system, causing the disease known as ‘crown gall’ that occurs in greenhouse and fruit crops7. Its QS system is homologous to LuxI /LuxR by Vibrio fischeri and is made up of the autoinducer synthase, the TraI protein, the N-(3-oxo-octanoyl)-L-homoserine lactone (3OC8HSL) autoinducer and the transcriptional receptor, the TraR protein5. This communication system is activated when the lactones synthesized by TraI at high population densities accumulate until reaching a certain level and when they reach that concentration threshold, they are detected by TraR. The TraR-3OC8HSL complexes activate the transcription of the target genes in the Ti plasmid, including those required for conjugation8. In effect, the TraR protein is a transcriptional regulator in the QS mechanism of A. tumefaciens.\n\nSince QS signaling is vital for the regulation of bacterial gene expression that has an impact on numerous microbial physiological functions, including those related to pathogenicity in humans, plants and animals, there is enormous interest in the design and implementation of QS system inactivation strategies9. One of the strategies for interruption of the QS is the blockade of the transduction of the QS signal, which can be achieved by the presence of an AHL antagonist capable of competing or interfering with the binding of the signal molecule to its LuxR receptor10.\n\nFor this purpose, we combined two molecular modeling strategies: molecular docking and three-dimensional quantitative structure-activity relationship (3D-QSAR). First, the molecular docking methodology was applied to a series of 34 analogues of AHL on the TraR transcriptional receptor to simulate the binding of analogues at the active TraR site. Secondly, analysis of the structural characteristics and interactions of the ligands was carried out in order to evaluate how the inhibitory activity is affected by its properties. Finally, 3D-QSAR models were generated to describe the correlation between the experimental biological activity using partial least squares (PLS) calculations and steric and electrostatic properties, which theoretically predict the activity of the 34 analogues of AHL through statistical parameters and evaluate the prediction of the models obtained.\n\n\nMethods\n\nThe chemical structures of 34 AHL analogues were taken together with their half maximal inhibitory concentration (IC50) biological activity from Geske et al. (Table 1)11,12. These structures were optimized using the theory of functional density with the functional X3LYP, together with the DGDZVP calculation base13,14, using the Gaussian09 program15 (as an open-source alternative, GAMESS could be used16).\n\nAHL, acyl homoserine lactone; PHL, phenylacetyl homoserine lactone; POHL, phenoxyacetyl homoserine lactone; PPHL, phenylpropionyl homoserine lactone. Structures and IC50 values for each analogue were obtained from Geske et al. 200711.\n\nMolecular docking calculations were performed to predict the binding modes among the 34 analogues of AHL and the TraR receptor. The 3D structure of the TraR receptor and its native co-crystallized ligand 3OC8HSL were downloaded from the Protein Data Bank, with the identifier 1L3L. The complex in PDB format was visualized and prepared using the structure preparation tool available in the Sybyl X 2.1.1 package17 and the Amber force field was used to assign the partial atomic charges of the protein. As an alternative, the open-source program, Avogadro could be used18. The native ligand and all water molecules were removed from the complex, hydrogen atoms were added and side chain amides and imidazoles were fixed (protonated), assuming a physiological pH of 7. The structure of extracted native ligand was optimized with the same conditions as the 34 analogues, mentioned above.\n\nOnce the structure of the native ligand and the TraR receptor was obtained, the docking protocol was initiated in the molecular simulation program AutoDock 4.219. The preparation of both the protein and ligand was performed using the graphical user interface of AutoDock, called AutoDockTools20. This preparation involved the addition of hydrogen and the assignment of Gasteiger charges21. After this, the potential maps for each type of atom in the ligand were calculated, executing the grid parameter using the Autogrid program incorporated in AutoDock. In this study, the grid box was delimited, centered on the TraR binding site, with dimensions 50x50x50 Å and spacing between points of 0.375 Å. Finally, for the conformational search, the Lamarckian genetic algorithm was chosen and for the validation 500 conformations were registered, with the remaining parameters used as per the default setting. With these parameters, docking validation was carried out by re-coupling the native ligand to the binding site of the TraR receptor.\n\nOnce the docking protocol was validated, and its ability to reproduce the interactions that promote protein-ligand binding confirmed for this particular case, TraR-3OC8HSL, the docking calculations for the 34 AHL analogues and the receptor in question were continued following the previous protocol, with the only difference being that 200 conformations were recorded for each analogue, of which the best were chosen according to their binding energy values and their position within the binding site of the TraR protein. The interactions of the selected protein-ligand complexes were analyzed on the Protein-Ligand Interaction Profiler webserver22.\n\nTwo 3D-QSAR models were constructed from structural alignments. The first model was constructed by taking the structure of the ligand, with the best inhibitory activity, reported by Geske et al. 200711, as a template. The other model was constructed based on the 34 best-selected poses of each analogue-TraR molecular docking23, which generated an automatic alignment24. The comparative molecular field analysis (CoMFA)25 descriptor was obtained by using the QSAR tool implemented in Sybyl X 2.1.1. Steric and electrostatic field energies were calculated by using the Lennard-Jones and Coulomb potentials, respectively, with a dielectric constant 1/r based on the distance of all interactions. To calculate the steric and electrostatic energies, we used the force field of Sybyl X.2.1.1, Tripos26. As an alternative, the open-source program, Open3DQSAR could be used27.\n\nFor the evaluation of the 3D-QSAR models obtained, the cross-validation technique was used with the ‘leave-one-out’ method. With this validation, the predictive power of the models was determined and, in this way, the best model was determined. An exploratory analysis of the correlation between molecular fields and biological activity was also performed using PLS regression; thus, the correlation coefficients (R2) of each model were obtained using the SAMPLS algorithm of Sybyl X 2.1.1. Finally, the cross-validation was done, giving the values of Q2 and R2CV. According to these values both models were compared, and it was deduced which had the best predictability.\n\n\nResults and discussion\n\nIn order to guarantee the reliability of the protocol used for molecular docking among analogues of AHL and the TraR receptor, validation of the search algorithm and scoring function combination was performed by re-docking the native ligand to the binding site of the TraR receptor using the molecular docking approach. From the large number of conformations generated, the conformations that best reproduced the position and interactions of the native ligand within the binding site were chosen. This conformation was compared with the co-crystallized structure of the same native ligand, as is depicted in Figure 1. In the alignment of the conformations of the native ligand in (green color) and the resulting docking (pink color), it can be seen that the coupled structure located at the TraR binding site adopts a conformation almost identical to the co-crystallized structure of the native ligand and in this way, both the lactone ring and the acyl side chain is located suitably at the receptor binding site.\n\nThe mean quadratic root deviation (RMSD) value calculated by AutoDock was used to confirm whether the chosen conformation was correct or not. The RMSD value of the chosen conformation is 0.56 Å; generally, an RMSD value of less than 1.5 or 2 Å is considered to be optimal28. In Figure 2, the interactions established in the conformation resulting from the molecular docking are visualized. In this ligand-receptor complex, the majority of hydrogen bond interactions that contribute to the binding of the native ligand and TraR are reproduced, as well as the correct folding of the TraR receptor29. These interactions include hydrogen bonding between the Trp 57 residue and the carbonyl group of the lactone ring, hydrogen bonding between Asp 70 and the amino group and a bond between the second carbonyl group and Thr 129, with the exception of the bond between the Tyr 53 residue and the first carbonyl group located in the side acyl chain. Hydrophobic residues also contribute to the stable binding of the native ligand with TraR by hydrophobic interactions with residues Leu 40, Tyr 53, Tyr 61, Val 72, Trp 85. The interactions with Thr 51, Phe 62 and Ile 102 do not occur in said conformation.\n\nThe structural interactions of this group of molecules were classified into five groups according to the differences present in the acyl chain reported by Geske et al.11,12. This classification was made taking into account the importance of the influence of the variation of the acyl chain on the interactions that form the 34 analogues of the AHL within the TraR binding site. In addition to the classification by groups, the individual structure of each analogue was divided into two regions, A and B, as is shown in Figure 3, the first consisting of the lactone ring and the carbonyl group and the second region comprising the acyl chain, which is the one that varies between the different analogues. In this way, the analysis of the interactions is facilitated since each analog tends to form mostly the same interactions in the region A, unlike those formed in region B, which tend to be different30.\n\nRegion A is formed by the lactone ring and the amide function and region B has variable side chains.\n\nFor each analogue-TraR docking, two or more clusters were generated. In these clusters, the different ways of binding can be identified. From the analysis of the clusters of each analogue-TraR docking, the best 34 conformations were chosen using the orientations that reported the lowest binding energy as the criterion of choice. The analogue-TraR complexes chosen, in comparison to 3OC8HSL, are, in general, similarly oriented at the TraR receptor binding site. The structure of these analogues was constructed based on the structure of homoserine lactone, with variations in the acyl chain of the native ligand. Table 2 shows the IC50 values and the free energies calculated using AutoDock for all 34 ligands.\n\nLigands 4, D6, C10, E22 and E35 have the highest values of inhibitory activity (IC50), so the analysis of their structure-activity relationship for each group was taken as precedents. Analogues 4, C10, E22 and E35 form two hydrogen bonds with the TraR receptor; one between the carbonyl group of the lactone ring and the amino group in the Trp 57 residue and the second between the carbonyl group of the first carbon of the acyl chain and the residue Thr 129. Ligand D6 also forms these two hydrogen bonds, but also forms another with the Asp 70 residue and the carbonyl group of the acyl chain. The native ligand forms the same three hydrogen bonds mentioned that contribute to the bond at the TraR protein binding site. Similarly, the remaining ligands form these bonds but with the absence of one or two of them and differences in bond distances. The interactions of ligands D6 and E35 are detailed in Figure 4.\n\nHydrogen bonds are shown in blue, hydrophobic interactions in gray, π-π stacking in green and halogen bonds in cyan.\n\nOn the other hand, the possible hydrophobic interactions formed by these ligands in the hydrophobic pocket of TraR involve both the lactone ring and the acyl chain. Structurally, ligand 4 is classified within the group of non-native AHLs with an acyl chain of seven carbon atoms; one less than the native ligand 3OC8HSL. This side chain allows the formation of eight possible hydrophobic interactions with Ala38, Leu40, Gln58, Phe62 and two interactions with each residue of Tyr61 and 63. In addition, in the lactone ring are the interactions with Val72 and Trip85, for a total of ten interactions. The D6 ligand has in its structure a side chain a carbonyl group on carbon 4 and a toluyl group. This structural modification places it within the group of other analogues, and between this chain and the protein it is possible to form 11 hydrophobic interactions; eight with the same residues as ligand 4 and also those formed with Ala49, Thr51 and Ala168. The C10 ligand has in its side chain a phenylacetanoyl group and E22 a phenoxyacetanoyl group, which places them in the phenylacetyl homoserine lactones and phenoxyacetyl homoserine lactones groups, respectively. Both form six hydrophobic interactions; four in the side chain with Leu40 and Tyr53 with different distances of connection, and two in the lactone ring with Val 72 and Trp85. Finally, of the 34 analogues, ligand E35 reported the highest inhibitory activity against TraR. It is classified in the phenylpropionyl homoserine lactones group since it has the phenylpropionyl group in its side chain and its possible hydrophobic interactions are the same as those mentioned for C10 and E22.\n\nThe differences among the possible hydrophobic interactions mentioned above for the different ligands representing each group and the interactions of the native ligand in the hydrophobic pocket of TraR are notable. However, these are not the only differences; some analogue-TraR complexes reported other possible interactions, such as π-π stacking, which are favored in ligands that in their structure possess an aromatic group, as can be seen in the complexes of ligands D6, C10, E22 and E35. The formation of this interaction occurs between the benzene rings of these molecules and the Tyr 61 residue. This type of interaction is also reported in other ligands, but with the amino acid Tyr53.\n\nOn the other hand, analogues that have halogen atoms in their side chains can form other types of interactions called halogen bonds. In this study, ligands C10, D15, E21, E23, B7, and E35 have halogen bond interactions with the Gln 58 residue and the E23 ligand with Trp 57. Studies of the characteristics of this type of halogen bond establish that they are strongly directional and the distance between the halogen and the nearby electronegative atom should ideally be less than the sum of its Van der Waals radii. Moreover, although the fluorine atom is a halogen, it does not have the capacity to form these interactions due to its great electronegativity31. According to the above, the halogen bonds of ligands D15, E23 and E35 are discarded as they are not possible since the interaction occurs between a fluorine atom and an oxygen atom. The sum of the van der Waals radii for the remaining ligands C10, E21 and B7 are calculated as follows: in the C10 ligand where its iodine atom and the Gln 58 oxygen interact, this value is 3.50 Å; in the E21 ligand where its bromine atom and the Gln 58 oxygen interact, the sum of the radii is 3.37 Å; and in ligand B7 where its bromine atom and oxygen of Gln 58 interact, the sum of the radii is 3.37 Å. The respective distances between the atoms bound for each ligand-receptor complex are 3.90 Å , 2.14 Å and 2.06 Å. Comparing these values, it follows that the only halogen bonds that are shorter than the value of the sum of the radii of the bound atoms are those of ligands E21 and B7 with the amino acid Gln 58.\n\nIt is quite clear that the addition of certain groups such as phenyl and halogenated substituents can generate other interactions, which could confer changes in the increase or decrease in the inhibitory activity of these analogues against the TraR receptor. These extra interactions (halogen bond, π-π stacking), formed by the analogues and the TraR receptor, are not present when TraR is in complex with the native ligand.\n\nFor ligand-based alignment, ligand E35 was used as a template since it was the ligand with the highest inhibitory activity against TraR and for the receptor-based alignment, the best poses generated in the molecular docking, that is, those that reported the lowest binding energy and the correct orientation in the active site of the receptor, were used. Figure 5A and 5B show the superposition of all ligands for the ligand-based model (Model 2) and receptor-based model (Model 1), respectively.\n\nA) Model 1 and B) Model 2.\n\nThe correlation analysis between CoMFA values and in vitro biological activities of the 34 analogues was performed by PLS and the evaluation of the predictive capacity of the models generated through the leave-one-out cross-validation method. Q2 values of 0.457 and R2 are obtained with cross validation of 0.856 for the Model 2, and a Q2 of 0.697 and R2 with cross validation of 0.781 for the Model 1. In effect, Model 1 shows a lower correlation so that their statistical confidence is lower than in model 2. Table 3 shows the other results of both PLS and cross-validation analyzes.\n\nSEE, standard error of estimate.\n\nFigure 6 shows correlation of models 1 and 2, it can be seen that in model 2 there is a better pattern of adjustment of the points along the trend line, which translates into the prediction of biological activity values closer to those reported experimentally, unlike the graph for Model 2 where the residual dispersion is greater. From these comparisons, the Model 2 is statistically profiled as the one with the best correlation and the greatest predictive power. Even so, the Model 1 should not be discarded altogether since it also takes into account the structural similarity between the analogues and the native ligand; the position and orientation taken by the ligands at the receptor binding site according to the conformational freedom allowed by the amino acid residues that comprise it.\n\nThrough the interpretation of the contour maps obtained by CoMFA, it is possible to know which structural characteristics of the ligands correlate with the changes in their biological activity values. These contours are represented by colors; steric contour maps are represented in green (region favorable for bulky groups) and yellow (region not favorable for bulky groups), while those of red (region favorable for electronegative substituents) and blue (region favorable for electropositive substituents) correspond to electrostatic contours32.\n\nIn Figure 7 the contour maps of the CoMFA models calculated are shown, taking as an illustrative example those generated for ligand D15, which records one of the highest values of inhibitory activity against TraR in the phenoxyacetyl homoserine lactones group. In these contours, it can be observed that the sterically favored regions are in positions close to the fluorine atoms found in the benzene ring substituent in the ligand structure in both Figure 7A and 7B. This is because this region has high electronegativity, which favors interactions with residues that have partially positive charges in their structures, while the sterically unfavorable regions, in yellow, spread around the phenyl group. However, on most of the hydrogen atoms in the phenyl ring, the positively charged regions that favor the electrostatic field are identified, as can be seen in more detail in Figure 7B. In the same way, carbonyl and ether groups with a negative charge are seen as electrostatically favorable regions due to the negative potential of oxygen atoms. In addition, the existence of halogens promotes the formation of halogen bonds with residues containing oxygen atoms in their structure. All these groups mentioned are identified as regions that would contribute to the antagonistic activity of ligand D15 against TraR.\n\nA) receptor-based model and B) ligand-based model. Steric field: green: sterically favorable; yellow: sterically unfavorable. Electrostatic field: red: negative potential favorable; blue: negative potential unfavorable.\n\n\nConclusions\n\nIn most of the 34 ligand-receptor complexes obtained by molecular docking, the formation of hydrogen bonds and similar hydrophobic interactions with the same amino acid residues were observed. The most common hydrogen bonds are established with Trp 57, Thr 129 and Asp 70. The most recurrent hydrophobic interactions are formed with Ala 38, Leu 40, Tyr 53, Tyr 61, Val 72, Trp 85. Certain structural modifications such as the presence of aromatic rings and halogen atoms allow the formation of other interactions such as molecular type π-π stacking and halogen bonds. From the above, it is possible to affirm that there is a marked relationship between the type of interactions and the activity of the ligands, where the differences with those formed by the native ligand may improve the inhibitory activity of the analogues against TraR.\n\nThe PLS statistical analysis of each model and subsequent internal validation allowed comparison of the predictive capacity under parameters such as R2 and Q2, analyzing how the predicted inhibitory activity values vary when the experimental biological activities are correlated with the properties of the optimized structures of the ligands (model based on the ligand) and, for the model based on the receptor, when the same experimental activities are correlated with the properties of the poses that the ligands adopt in the molecular docking. This resulted in the best model in statistical terms being the model based on the ligand. Even so, the receptor-based model, since it is based on the simulations of the analogue receptor binding, provides information based on the position and orientation that the analogues can adopt according to the chemical nature of the TraR receptor's active site, unlike the model based on the ligand, which is based solely on optimized structures.\n\nRegarding the study of the relationship of the structural characteristics that influence the values of biological activity, the 3D-QSAR-CoMFA models suggest that it is possible to improve the inhibitory activity of new ligands by adding groups with positive and negative charges of favorable electrostatic potentials such as carbonyl groups, ether groups, halogens, and in some cases the nitro substituents and the phenyl group, which favors the formation of hydrophobic interactions. Bulky groups attached to the phenyl ring and other groups such as sulfur are identified as unfavorable regions for the design of new analogues with greater inhibitory activity against TraR.\n\n\nData availability\n\nFigshare: Receptor_Alim_2019_Pose_E_D_1.mdb (1).zip. https://doi.org/10.6084/m9.figshare.10255739.v123.\n\nThis project contains the following underlying data:\n\n- .mol2 files (templates used for alignment in MOL2 format)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nMAM and RVR thank the Universidad de Cartagena (Cartagena, Colombia) and Fundación Universitaria Tecnológico de Comfenalco for continuous support to the group.\n\n\nReferences\n\nKuttler C, Hense BA: Interplay of two quorum sensing regulation systems of Vibrio fischeri. J Theor Biol. 2008; 251(1): 167–180. PubMed Abstract \| Publisher Full Text\n\nBarreto AC: Quorum Sensing: Sistemas de comunicación bacteriana. Ciencia Actual. 2013; 5–6.\n\nLang J, Faure J: Functions and regulation of quorum-sensing in Agrobacterium tumefaciens. Front Plant Sci. 2014; 5: 14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMattmann ME, Blackwell HE: Small molecules that modulate quorum sensing and control virulence in Pseudomonas aeruginosa. J Org Chem. 2010; 75(20): 6737–6746. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWaters CM, Bassler BL: Quorum sensing: cell-to-cell communication in bacteria. Annu Rev Cell Dev Biol. 2005; 21: 319–346. PubMed Abstract \| Publisher Full Text\n\nPedroza CJ: Caracterización de la enzima acil homoserina lactonasa de una cepa de bacillus thuringiensis. Medellin: Universidad nacional de Colombia. 2010. Reference Source\n\nAlippi A, Lopez A, Balatti P: Métodos para la detección de Agrobacterium a partir de muestras de material vegetal, suelo y agua. Revista Argentina de Microbiología. 2011; 43(4): 278–286. Reference Source\n\nLang J, Faure D: Functions and regulation of quorum-sensing in Agrobacterium tumefaciens. Front Plant Sci. 2014; 5: 14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDong YH, Wang LH, Zhang LH: Quorum-quenching microbial infections: mechanisms and implications. Philos Trans R Soc Lond B Biol Sci. 2007; 362(1483): 1201–1211. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrtero MR: Revista real academia Galega de ciencias. 2010; XXIX: 6–7. Reference Source\n\nGeske GD, O´Neill JC, Miller DM, et al.: Modulation of Bacterial Quorum Sensing with Synthetic Ligands: Systematic Evaluation of N-Acylated Homoserine Lactones in Multiple Species and New Insights into Their Mechanisms of Action. J Am Chem Soc. 2007; 129(44): 13613–13625. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGeske GD, Mattmann ME, Blackwell HE: Evaluation of a focused library of N-aryl L-homoserine lactones reveals a new set of potent quorum sensing modulators. Bioorg Med Chem Lett. 2008; 18(22): 5978–5981. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGodbout N, Salahub DR, Andzelm J, et al.: Optimization of Gaussian-type basis sets for local spin density functional calculations. Part I. Boron through neon, optimization technique and validation. Can J Chem. 1992; 70(2): 560–571. Publisher Full Text\n\nXu X, Zhang Q, Muller RP, et al.: An extended hybrid density functional (X3LYP) with improved descriptions of nonbond interactions and thermodynamic properties of molecular systems. J Chem Phys. 2005; 122(1): 14105. PubMed Abstract \| Publisher Full Text\n\nFrisch MJ, Trucks GW, Schlegel HB, et al.: Gaussian 09. Wallingford CT: Gaussian, Inc. 2009. Reference Source\n\nGordon MS, Schmidt MW: Advances in electronic structure theory: GAMESS a decade later. In \"Theory and Applications of Computational Chemistry: the first forty years\". Dykstra CE, Frenking G, Kim KS, Scuseria GE, (editors), Elsevier, Amsterdam, 2005; 1167–1189. Publisher Full Text\n\nTRIPOS Associates, Inc: Sybyl-X Molecular Modeling Software Packages, Version 2.0. St. Louis, MO USA. 2012. Reference Source\n\nHanwell MD, Curtis DE, Lonie DC, et al.: Avogadro: an advanced semantic chemical editor, visualization, and analysis platform. J Cheminform. 2012; 4(1): 17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMorris GM, Goodsell DS, Pique ME, et al.: AutoDock Version 4.2. Citeseer: 2012; 1–6. Reference Source\n\nHuey R, Morris G: Using AutoDock 4 with AutoDockTools: A Tutorial. The Scripps Research Institute, USA: 2008; 1–56.\n\nMorris GM, Huey R, Olson AJ: Using AutoDock for ligand-receptor docking. Curr Protoc Bioinformatics. 2008; 24: Chapter 8:Unit 8.14. PubMed Abstract \| Publisher Full Text\n\nSalentin S, Schreiber S, Haupt VJ, et al.: PLIP: fully automated protein-ligand interaction profiler. Nucleic Acids Res. 2015; 43(W1): W443–W447. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVivas-Reyes R: Receptor_Alim_ 2019_Pose_E_D_ 1.mdb (1).zip. figshare. 2019. http://www.doi.org/10.6084/m9.figshare.10255739.v1\n\nSippl W: Development of biologically active compounds by combining 3D QSAR and structure-based design methods. J Comput Aided Mol Des. 2002; 16(11): 825–830. PubMed Abstract \| Publisher Full Text\n\nCramer RD, Patterson DE, Bunce JD: Comparative molecular field analysis (CoMFA). 1. Effect of shape on binding of steroids to carrier proteins. J Am Chem Soc. 1988; 110(18): 5959–5967. PubMed Abstract \| Publisher Full Text\n\nClark M, Cramer RD, Van Opdenbosch N: Validation of the general purpose tripos 5.2 force field. J Comput Chem. 1989; 10(8): 982–1012. Publisher Full Text\n\nTosco P, Balle T: Open3DQSAR: a new open-source software aimed at high-throughput chemometric analysis of molecular interaction fields. J Mol Model. 2011; 17(1): 201–208. PubMed Abstract \| Publisher Full Text\n\nHevener KE, Zhao W, Ball DM, et al.: Validation of molecular docking programs for virtual screening against dihydropteroate synthase. J Chem Inf Model. 2009; 49(2): 444–460. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang RG, Pappas KM, Brace JL, et al.: Structure of a bacterial quorum-sensing transcription factor complexed with pheromone and DNA. Nature. 2002; 417(6892): 971–974. PubMed Abstract \| Publisher Full Text\n\nAhumedo M, Drosos JC, Vivas-Reyes R: Application of molecular docking and ONIOM methods for the description of interactions between anti-quorum sensing active (AHL) analogues and the Pseudomonas aeruginosa LasR binding site. Mol Biosyst. 2014; 10(5): 1162–1171. PubMed Abstract \| Publisher Full Text\n\nSaavedra LH: Entendiendo los puentes de halógeno desde un punto de vista teórico. Santiago de Chile: Universidad de Chile. 2013. Reference Source\n\nNair PC, Sobhia ME: CoMFA based de novo design of pyridazine analogs as PTP1B inhibitors. J Mol Graph Model. 2007; 26(1); 117–123. PubMed Abstract \| Publisher Full Text" }	[ { "id": "58629", "date": "24 Jan 2020", "name": "Nestor Cubillan", "expertise": [ "Reviewer Expertise Computational Chemistry", "Mathematical Chemistry", "Chemometrics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this work, the authors proposed a dataset for studying interaction of a set of 34 molecules with the Agrobacterium tumefaciens TraR binding site through Molecular Docking and 3D QSAR. The work is well written and readable. I have an unique concern about the ligand-based ComFA model results in table 3. The model 1 presents an R2 of 0.781 while Q2 is 0.697 revealing good robustness of this Model. However, Model 2 has an R2 of 0.856 and Q2 of 0.457. This result is evidence of the presence of data leveraging the model. Can the authors interpret this behaviour?\nAn explanation of this result can be related to noisy variables in predictors overfitting the model. Is this a possible explanation of these results?\nI considered this work suitable for publication after making these minimum corrections.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5205", "date": "18 Feb 2020", "name": "Ricardo Vivas-Reyes", "role": "Author Response", "response": "Dear Sir,This is the answer to the reviewer, where we give the reason for the two models used in this study. Also waiting for the referee to agree with our response. Thank you in advance for your time and dedication.The idea of the two models was realized to compare the predictive capacity of both models. So, a good value for Q2 is a value close to R2. That means that your PLS model works independently of the specific data that was used to train the PLS model, which is the case of model 1, while inconsistencies occur in the model.Thanks in advanceBest Regards,Ricardo" } ] }, { "id": "58098", "date": "10 Mar 2020", "name": "Leonardo C. Pacheco-Londoño", "expertise": [ "Reviewer Expertise Chemometrics", "intelligence artificial", "sensor and biosensor design", "material science", "vibrational spectroscopy", "and ab-initio calculation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors try to explain the interactions among structural analogues of acyl homoserine lactones (AHLs) using molecular modeling and support this by a statistical correlation of biological activity with molecular descriptors. The problem of the article is the type of linear model used for the statistical correlation. PLS is a type of correlation and this generates multiple linear correlations. These are known as latent variables and these are the variables that are not directly observed but are inferred (through a mathematical model) from other observed variables or (molecular descriptors). In this case, this type of model is viable when you have many samples (molecules) and many independent variables (molecular descriptors), and this is not the case. In my opinion for the article to be published the authors must include the following:\nYou must indicate how many descriptors are used in the models.\n\nThey use 34 molecules; they should remember that the number of degrees of freedom (g) for PLS models would be 34 - # LV- # descriptors-1. The authors do not report the number of latent variables (LV). If the g is minimal, the PLS model must be discarded.\n\nThe authors must realize an analysis of the LV. This should be showing the weights or loading for each LV, and thus indicate which descriptors are contributing to each LV and be able to explain: (1) why the correlations should be independent based on the weights of the descriptors in the LVs and (2) why these descriptors do not work well In a single multiple linear equations and it is necessary to generate several linear combinations (LV) to find a good correlation.\n\nIf the model has only one descriptor or two, the PLS process of adding LV to improve the correlation generates what is known as overfitting though that a leave-one-out cross-validation is being used. They should better perform a multiple regression for this case.\n\nMost likely, the authors have already tried multiple regression and found no correlation. If this is the case, there is a high probability that there is no true correlation between the data of the descriptors obtained and the biological activity. I invite you to improve the calculation of these descriptors.\n\nThe authors must include an external validation in addition to the cross-validation\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-2062
https://f1000research.com/articles/8-1732/v1	09 Oct 19	{ "type": "Research Article", "title": "Epitope mapping of an uncertain endogenous antigen implies secretogranin II peptide splicing", "authors": [ "David R. Howlett", "Iain J. Clarke", "Russell P. Newton", "John E. Hart", "Iain J. Clarke", "Russell P. Newton", "John E. Hart" ], "abstract": "Background: The search for a tissue-mass reducing reproductive hormone involved a bioassay-guided physicochemical fractionation of sheep blood plasma. This brought forth a candidate protein whose apparent mass on gels and in mass spectrometry (MS) was 7-8 kDa, implying a polypeptide of ~70 residues. Four purification runs gave Edman N-terminal sequences relating to 1MKPLTGKVKEFNNI14. This is bioinformatically obscure and has been resistant to molecular biological investigation. The sequence was synthesized as the peptide EPL001, against which was raised a goat polyclonal antiserum, G530. Used in an antigen capture campaign, G530 pointed to the existence of a novel derivative of secretogranin II (SgII), the neuroendocrine secretory vesicle helper protein and prohormone. The proposed SgII derivative was dubbed SgII-70, yet the sequence commonality between SgII and EPL001 is essentially NNI. Methods: Immunohistochemical (IHC) labelling with G530 is reported within rat, mouse and human cerebrovasculature and in glandular elements of the mouse intestine. Epitope mapping involved IHC peptide preabsorption, allied to deductive bioinformatics and molecular modelling in silico. Results: G530 is deemed monoepitopic in regard to both its synthetic antigen (EPL001) and its putative endogenous antigen (SgII related). The epitope within EPL001 of the anti-EPL001 antibody is inferred to be the contiguous C-terminal 9KEFNNI14. This is so because the G530 blockade data are consistent with the epitope in the mammalian endogenous antigen being part contiguous, part non-contiguous KE·F·NNI, ex hypothesi. The observed immunostaining is deduced to be due to pre-SgII-70, which has a non-C-terminal NNI, and SgII-70, which has an N-terminal MLKTGEKPV/N and a C-terminal NNI (these two motifs being in the reverse order in the SgII parent protein). Conclusion: The present data are consistent with the hypothesis that the anti-EPL001 antibody binds to an SgII-related epitope. SgII is apparently subject to peptide splicing, as has been reported for the related chromogranin A.", "keywords": [ "Antibodies", "Antigens", "Peptides", "Epitopes", "Bioinformatics", "Proteomics", "Imaging" ], "content": "Introduction\n\nPolyclonal antisera (hereafter ‘antibodies’) raised in rabbits and a goat to a synthetic peptide have provided neuroendocrine immunohistochemistry (IHC) images in mammals (human, sheep, rat) and in the embryo of the fruit fly Drosophila melanogaster1. But what exactly were the antibodies seeing endogenously? They had been raised as a result of a hunt for a tissue-mass inhibiting reproductive hormone2. A peptide of 7-8 kDa (polyacrylamide gel electrophoresis, MALDI-TOF MS) was found in sheep jugular vein plasma subjected to fractionation via ultrafiltration and guided by assays in vivo (internal organ reduction in rats) and in vitro (reduced division of rat bone marrow cells), but scant amino acid sequence data could be obtained before the target molecule was lost to view. In this prior work1 an unambiguous sequence obtained by automated step-wise Edman degradation (Applied Biosystems/PROCISE, Foster City, CA, US) was the N-terminal 14 amino acids MKPLTGKVKEFNNI, synthesized as a peptide designated EPL001. The preceding purification run provided the partially similar ML/KPLTGQAMEF, while a following run delivered the highly similar MKPLT/GKVKxFNNI. On another occasion readings at only four positions could be obtained, providing, however, in-register matches: - - P - - - - V - - FN. This was deemed significant as it involved a maximally purified bioactive fraction derived from ultrafiltered sheep plasma subjected also to gel filtration and anion exchange chromatography. The unambiguous 14-residue sequence is bioinformatically obscure and proved resistant to investigation by molecular biology approaches involving the use of oligonucleotide probes and RT-PCR to find matching sequences in DNA and RNA libraries and the use of anti-EPL001 antibodies to identify cDNA synthesized proteins. The 14 residues MKPLTGKVKEFNNI were synthesized as a peptide designated EPL001. A goat anti-EPL001 antibody was raised1 and designated G530. Apart from being used in IHC, G530 was deployed in an antigen capture campaign featuring immunoprecipitation (IP) with liquid chromatography-mass spectroscopy (LC-MS), using two main feedstocks: aqueous extract of rat hypothalamus and fruit fly embryo material1. The former was tested for bioactivity. It proved to have anti-proliferative and pro-apoptotic effects in an assay in vitro involving rat bone marrow cells. This inhibitory influence was subject to prior immunodepletion by an anti-EPL001 antibody, except when peptide EPL001 was added as well during the immunodepletion process, achieving preabsorption. Multiple lines of evidence indicated that the mammalian antigen was likely to be a proteoform of secretogranin II (SgII), the neuroendocrine secretory vesicle helper protein and prohormone. At ~70 residues, this polypeptide derivative was dubbed SgII-70 (pronounced ‘sig two-seventy’). Cryopreserved material in IP/LC-MS delivered no credible candidate. ‘Likely SgII relatedness’ arose from rat hypothalamic aqueous extract subjected to formalin fixation and antigen retrieval. What works in IHC seems to have worked in IP/LC-MS. Meanwhile the fruit fly antigen appeared to be an uncharacterised protein, (UniProt ID Q9W2X8), which was newly recognised as having extensive homology in detail with SgII. Both the fly and mammalian candidates bear homologues of the other’s MS ID peptide1.\n\nThe G530 IP/LC-MS campaign yielded a protein identified by the MS software as Q8CGL8, a splice variant of rSgII of 37.1 kDa, though it could equally have been full-length rSgII. This was the only item snare in both of the two forms of antigen retrieval used and so was to that extent the sole mammalian candidate, but SgII-70 itself was not bagged. In regard to EPL001, preliminary mapping of the epitope – defined as that part of an antigen molecule to which an antibody binds – involved dot blot analysis with G530 of three peptides: full length EPL001 and two component peptides, the N-terminal 1MKPLTG6 and the C-terminal 7KVKEFNNI141. This showed that the synthetic epitope (singular for parsimony) resides in the C-terminal section of EPL001. Mammalian Q8CGL8 has the EPL001 C-terminal match V---NNI, while fly Q9W2X8 has K----NNI, sketchy resemblances both. Is the EPL001 sequence really related to these proteins?\n\nA chance observation provided a platform for the current investigation, which amounts to an attempt to get beyond the frustrating vagaries of purification and instead use G530 in IHC to elucidate the primary sequence of whatever it is that the antibody sees endogenously, putatively SgII-70. SgII has a pair of domains which sort the protein intracellularly into secretory vesicles3. It was noticed that the ovine-derived EPL001 sequence, MKPLTGKVKEFNNI, finds a nine-residue resemblance in the second sorting domain of sheep SgII (W5QEU8). The nine-residue string, ‘sSgII-9’, is 367MLKTGEKPV375 (residue numbering with signal sequence). The shaded residues match those from the front half of EPL001, in the form of three doublets, separated by singleton matches to the residues in the second half of EPL001. Disregarding the apparent non-random interleaving of the front and back halves of EPL001, Spearman’s rank correlation between sSgII-9 and EPL001 is 0.59, a moderate positive correlation. The probability of a nine-residue partial anagram of EPL001’s 11 residues (i.e. minus NNI) occurring by chance in a typical ovine protein is about 1 in 146,000, as previously calculated1. Going further, EPL001 and sSgII-9 have the same initial residue: methionine. The probability of this is 1 in 11. The overall likelihood therefore of there being a methionine-commencing nine-residue anagram is 1/146000 x 1/11 or about 1 in 1.6m. The likelihood of there being a nine-residue anagram and NNI in the same protein is tinier still. (NNI probably has sorting domain relatedness too1, like sSgII-9.) Finally, there is an alignment of M - - - - - K between EPL001, sSgII-9 and the homologue of SgII’s second sorting domain within fly Q9W2X8 that is also unlikely to be due to chance. Why EPL001 might be an encoded version of sSgII will be considered later (see Discussion). Comparing EPL001’s C-terminal section with sSgII sequence elements sets up the prediction that the endogenous epitope could involve six residues, thus:\n\n\n\nOr thus, reading sSgII-9 in reverse:\n\n\n\nThese possibilities can be represented as K·E·F·NNI and KE·F·NNI, respectively. There are numerous other combinations of three or more residues from these sSgIIs sequence elements that match the order of residues in EPL001’s C-terminal section, such as K·V·F·NNI and V·KE·F·NNI. All are mixed, i.e. part contiguous, part non-contiguous, except NNI.\n\nThis paper attempts to deduce the endogenous epitope of the G530 anti-EPL001 goat antibody in mammals, via IHC peptide preabsorption studies on selected tissues (cerebrovasculature, gut), aided by deductive bioinformatics and molecular modelling in silico. Preabsorption, i.e. mixing of the antibody with antigen prior to application of the antibody, to block staining, has been achieved in western blotting with the C-terminal EPL001 peptide but not with the N-terminal peptide, in regard to aqueous extract of rat hypothalamus purified using an immunoaffinity column1. Both the western blotting and the immunopurification used G530. IHC is not described in a review of epitope mapping methods4, but is comparable to ELISA-based peptide-panel techniques for dissecting antigen-antibody interactions. The hypothesis here is that the G530 anti-EPL001 antibody binds to a SgII-related epitope; the null hypothesis is that it binds to something else. The hypothesis informed preabsorption peptide design and predicts that the endogenous epitope is probably a part non-contiguous version of EPL001’s presumed contiguous epitope. Data consistent with the SgII hypothesis and its epitope prediction are presented herein. This first attempt to elucidate the primary sequence implies that SgII-70 is the product of peptide splicing.\n\n\nMethods\n\nThe goat anti-EPL001 antibody was chosen for this IHC investigation because it had been used with success in the antigen capture campaign1 to disclose the target molecule’s apparent relatedness to SgII. Prior published IHC images have, however, been obtained predominantly using rabbit antisera, preferred in this application. The goat polyclonal antiserum (G530) was raised as described elsewhere1,5. A cysteine EPL001 peptide was synthesized conjugated at its N-terminus with the carrier protein KLH. The goat was injected simultaneously with antigen (400 µg) in PBS mixed with an equal volume of complete Freund’s adjuvant followed by eight booster injections at monthly intervals. An antibody dilution curve was obtained1. Titre was also established via IHC, with blockade of rat and ovine hypothalamic staining by EPL001 at 0.5 mg/ml. This and other examples of IHC preabsorption have been described previously1. No staining was seen with pre-immune serum. An antibody to LRP1 (ABP-PAB-10774) was obtained from a commercial supplier (Allele Biotech, San Diego CA), as was an antibody to SgII (ab192824, rabbit polyclonal to chromogranin C/SgII, raised to a recombinant fragment within human chromogranin C/SGII aa 1–277; Abcam UK).\n\nPeptides for use in IHC competition studies were synthesized by a commercial supplier (Peptide Protein Research Ltd, Fareham, UK). The peptides were manufactured to GLP using Fmoc solid phase synthesis. Purification involved RP-HPLC using water and acetonitrile as the mobile phases. Peptides were then analysed via LC-MS to determine mass and purity. All peptides were stored at -20°C prior to use. Amino acid sequences are given in Table 1, together with notes on provenance. A control peptide was deployed, in the form of EPL030. This is a random scrambling of the amino acid sequence of EPL001. Peptide design was informed by an earlier analysis1, expanded upon in Introduction, which conjectures that the EPL001 sequence can be decoded to reveal SgII.\n\nAmino acid sequences, proprietary codes, species background, relationship to EPL001 or SgII and ability to preabsorb in IHC the anti-EPL001 antibody G530. Peptides were used at 10 μg/ml (vs G530 at ~1 μg/ml).\n\nBrain, small intestine, kidney and spleen tissue was obtained from mice (six C57/Bl6 male mice, supplied by Charles River Laboratories, approximately six months old and weight 35–45g). Mice were housed with free access to Global Rodent Maintained Diet (Harlan Teklad) and water. They were maintained in an ambient temperature of 21±1°C under a controlled light–dark photocycle (12:12 h), with lights on at 07:00 h. Mice were humanely euthanised by overdose of sodium pentobarbitone). Brain tissue was also obtained from rats and humans. The rat details are as follows: four male Wistars supplied by Charles River Laboratories, six months old, 125–150 g; housed under a 12h light/dark cycle with ad libitum diet (Global Maintained Diet, Harlan Teklad); euthanasia via a sodium pentobarbitone overdose. The human details were thus: post-mortem cortex, aged control subjects, two male and two female, age 76–87 years; see Ethics. Formalin-fixed, wax-embedded blocks, cut into 7-μm sections and mounted onto slides, were used for IHC. Mounted sections of cerebral cortex from bovine brain were obtained from AMSBIO Biotechnology, Abingdon, OX14 4SE. Sections were dewaxed and rehydrated using Histoclear and alcohol dilutions. Antigen retrieval was carried out by microwaving the sections for ten minutes in citrate buffer pH 6.0. Following blocking of endogenous peroxidases (0.3% H2O2 in PBS for 30 minutes, for DAB sections only), sections were incubated overnight with primary antibody at a dilution of 1:4000. In initial preabsorption experiments, G530 was preincubated with a ten-fold excess of competing peptide for 30 minutes, before being added to the sections. Peptides were used at 10 μg/ml (vs G530 at ~1 μg/ml). Development of the sections was performed using biotinylated secondary antibodies at a 1:500 dilution (BA1000/RRID AB_2313606; BA-2000/RRID AB_2313581; BA5000/RRID AB_2336126), ABC reagents (PK-6100/ RRID AB_2336819, used according to manufacturer’s instructions) and a DAB kit (all Vector Laboratories, Peterborough, UK). Sections were briefly counterstained with Mayer’s hematoxylin solution before dehydration, mounting with DPX and coverslipping. For control experiments, the secondary biotinylated antibody was omitted. In some experiments, following incubation with G530 (± competing peptide), the secondary antibody was anti-goat Alex Fluor 568 used at a 1:500 dilution (A11079/AB_2534123; Invitrogen, ThermoFisher Scientific, Waltham, MA, USA).\n\nG530 was preabsorbed with tripeptides based on the epitope-relevant C-terminal section of EPL001. This was with and without the EPL001 parent peptide. The aim of the ‘with EPL001’ protocol was to block EPL001’s previously demonstrated inhibition of IHC staining.\n\nIn an IHC dose-response study involving the EPL001 C-terminal tetrapeptide FNNI and alanine substituted versions thereof the tissue consisted of serial sections of mouse small intestine and coronal sections of rat brain. The G530 antibody (final dilution 1:4000) was incubated in primary incubation buffer (PBS + 0.3% Triton X-100 + 2% bovine serum albumin) with peptide (dissolved in high purity water) at dilutions between 0.1 ng/ml and 1 µg/ml (final peptide concentration) for 30 min prior to addition to sections (tissue dewaxed and rehydrated; antigen retrieval with citrate at pH 6.0). Sections were incubated overnight at 4ºC. Further development was with the anti-goat secondary antibody Alexa Fluor 568. Serial images of matching features, either within the walls of small cerebral blood vessels or in glandular elements of the mouse small intestine, were analysed using ImageJ version 1.52i with a fixed threshold to give a value for ‘area labelled’.\n\nModels in silico were developed using Molecular Modelling Pro Plus, version 6.22, and ChemSite, version 5.10, produced by ChemSW (Accelrys Inc., San Diego, USA; Avogadro is an open-access alternative). Models were constructed by sequential additions of amino acid residues. Each model was adjusted in conformation to minimize energy levels: energy minimization was carried out in 1-fs time steps, to a total of 10,000 fs, with 100 equilibrium steps per iteration. Iterations were continued until six repeat iterations yielded no change in energy gradient. Analysis of interatomic distances mostly involved atoms in amino acid side-chains. Distances between pairs of atoms were computed automatically after atoms were selected manually on-screen. Each measurement was repeated twice more after closing the model and reloading to verify the initial measurement. For EPL001’s 9KEFNNI14, nine atoms were selected from side chains and two from the peptide backbone (Figure 6). This permitted 46 measurements, each atom to every other atom: a-b, a-c etc. Distances between the same atoms were calculated for KEFNNI as a free peptide, with comparisons reducing in number for free EFNNI through free FNNI to free NNI. Other ad hoc interatomic measurements are described in Results.\n\nCalculations to provide p values in the IHC image analysis were conducted using unpaired t-tests. A chi-squared test was used for interatomic distance comparisons in the molecular modelling, with measurements from the EPL001 model in silico representing expected (E) interatomic distances and measurements from the modelled free peptides (KEFNNI, EFNNI, FNNI, NNI) as observed (O) distances. Chi-squared values were calculated on the basis of (O-E)2/E.\n\nAll experimental procedures were conducted in strict compliance with applicable laws, regulations, rules and professional standards, with appropriate ethical oversight. The G530 antiserum was raised in compliance with the Australian Prevention of Cruelty to Animals Act 1986, with procedures approved by the relevant Animal Ethics Committee. The provision of animal tissue for histology in the UK was licensed in accordance with the Animals (Scientific Procedures) Act 1986. Human post-mortem tissue sections were provided, with ethical approval, by courtesy of Brains for Dementia Research Network (Alzheimer’s Society and Alzheimer’s Research UK). Data integrity has been maintained throughout, without outlier exclusions and with appropriate recording and archiving.\n\n\nResults\n\nAntibody G530, raised to the 14mer peptide EPL001, demonstrated labelling within the walls of cerebral blood vessels in mouse, rat and human, in a manner not previously described. The labelling was observed in the walls of arteries and arterioles, but not capillaries and appeared to be associated with the fibroblast and smooth muscle layers surrounding the contractile vessels (Figure 1). Co-labelling employing an antibody to LRP1 confirmed the vascular G530 labelling to be within blood vessel walls (Figure 2). In mouse small intestine sections, G530 produced labelling within the muscularis mucosae and possibly some columnar epithelium but no labelling within lamina propria (Figure 3), consistent with prior findings1. In other tissues evaluated prior to the study proper, labelling of blood vessel walls was observed within mouse spleen and kidney and bovine (this paper) and ovine brain1. Labelling was noted within cortical neurons of the species examined, with preabsorption in one series by EPL001 but not in another. The greater reliability of the cerebrovascular staining commended this as a focus in the present study. An antibody to SgII did not produce any labelling of mouse, rat and human cerebral blood vessels; although labelling was observed in the mouse small intestine, this bore no relation to G530 labelling. Raw images used to generate Figure 1–Figure 5 and Table 1 are available as Underlying data6–14.\n\nSections of human cingulate cortex were labelled by antibody G530 as described in Methods. (A–D) Cerebrovascular wall labelling at differing magnifications (scale bars: A = 500 μm; B, C = 25 μm; D = 10 μm). (E) Lack of labelling (arrowed) in cerebral capillary walls (scale bar = 500 μm).\n\nSections of human cingulate cortex were labelled by antibody G530 as described in Methods. (A) Labelling by an antibody to LRP-1. (B) Labelling by G530. (C) Merge of (A) and (B). Scale bars, 50 μm.\n\nWax sections of cerebral cortex, human (A, B) and mouse (C, D), and of mouse small intestine (E, F) were incubated with either antibody G530 (A, C, E) or G530 preabsorbed by a ten-fold excess of cognate peptide EPL001. Scale bars: (A–D) = 100 μm; (E, F) = 25 μm.\n\nIn initial peptide competition experiments, labelling within both human and mouse cerebral blood vessels and mouse small intestine was prevented by preincubating G530 with its cognate peptide EPL001 (Figure 3). Table 1 shows that peptides with a C-terminal NNI block labelling. Thus, effective blockers, in straightforward competition with the native antigen for antibody binding, are the NNI-concluding 14mers EPL142 and EPL143 (Figure 4), MKPVFNNI and the EPL001 C-terminal fragments KEFNNI, EFNNI and FNNI. Ineffective are six peptides: the fly and rodent SgII 20mer homologs and the 20mer extended form of EPL142 (EPL122), all with mid-sequence NNIs; a scrambled-sequence version of EPL001 lacking NNI but with a mid-sequence NI, KLKMNGKNIEPVFT; and two peptides lacking NNI altogether but terminating in N, in one case FN: MLKTGEKPN and MKPVFN. NNI was the only one of seven EPL001-related trimers that preabsorbed G530. Given this result, the attempt was not made to co-administer NNI and EPL001, inhibitors both. The other six trimers co-administered separately with EPL001 were ineffective as counter-inhibitors. EPL001 administered alone achieved near-total preabsorption across a range of concentrations (mean ± SEM, n = 4): 0.1 ng/ml = 0.56 ± 0.56; 1 ng/ml = 0.30 ± 0.30; 10 ng/ml = 0 ± 0; 100 ng/ml = 0.09 ± 0.05; and 1 µg/ml = 0 ± 0. Preabsorption titration across the same range of concentrations then focussed on FNNI, EPL001’s C-terminal tetramer, via alanine substitutions. Three tetramers in the form F - - I returned concentration-response curves; two tetramers in the form - - - I and F - - - did not (Figure 5). Labelling was actually increased by ANNI, except at the highest concentration. Preabsorption with EPL001’s C-terminal tetramer FNNI was asymptotic at the highest concentrations, with 2.7% staining (G530 alone bring 100%) at 100 ng/ml (~0.2 μM/L) and 3.5% at 1 μg/ml. Preabsorption was further investigated using EPL001’s C-terminal hexamer KEFNNI. At 100 ng/ml (~0.13 μM/L) the area of staining (as % of G530 staining without competing peptide) was 29.53 ± 9.69 (n = 8, p = 0.062, not significant (NS) vs preabsorption by EPL001 at 100 ng/ml), while at 10 μg/ml it was 4.7 ± 2.4 (n = 3, p = 0.12, NS). (The corresponding figures for MKPVFNNI were 44.40 ± 11.85, n = 8, p = 0.028 vs EPL001 and 3.92 ± 1.97, with n = 3, p = 0.11, NS.) In approximate terms, the IC50 preabsorption trend is as follows: EPL001 < 0.1 ng/ml; FNNI ≤ 10 ng/ml; KEFNNI ≤100 ng/ml.\n\nInteratomic distances between residues of different peptide sequences\n\nG530 was preincubated for 30 min with a x10 excess of potential preabsorption peptide before application to wax sections of mouse small intestine. (A) G530 alone. (B) G530 + EPL001. (C) G530 + EPL030. (D) G530 + EPL142. (E) G530 + EPL143. Amino acid sequences of peptides are shown in Table 1. Scale bars, 10 μm.\n\n(A) G530 (dilution 1:4000) was incubated for 30 min with tetramers at concentrations of 0.1 to 1000 ng/ml before application to the sections for IHC. Graph shows staining intensity (ImageJ) expressed as a percentage of G530 signal in absence of tetramer. Consistency of results invited data aggregation (murine gut, rat cerebrovasculature). Data are expressed as mean ± SEM of four determinations. The horizontal axis effectively represents the full preabsorption achieved with EPL001 at all concentrations. (B) Statistical comparisons (p values) are shown in the matrix for data points at 100 ng/ml (~0.2 μM).\n\nThe interatomic distances calculated via molecular modelling (Figure 6) yielded on statistical analysis a proportion of (O-E)2 values >10, betokening a big difference between observed and expected, as follows: KEFNNI, 39%; EFNNI, 11%; FNNI, 5%; NNI, 25%. Free FNNI is indistinguishable from EPL001’s 11FNNI14 in statistical terms (Chi2 = 4.54, degrees of freedom (dof) = 19, 99.5% confidence level). The null hypothesis, that there is no correlation between EPL001 and, separately, KEFNNI (Chi2 = 111.31, dof = 45, NS), EFNNI (Chi2 = 23.67, dof = 35, NS) or NNI (Chi2 = 4.36, dof = 7, NS), was accepted for the other comparisons of interatomic distances (see Underlying data)15. Interatomic distances were also compared between the Ks and E in the C-terminal section of EPL001 (7KV9K10EFNNI) and those in sSgII-9: ML3KTG6E7KPV (see Introduction). The same side chain measurements (in Å) were made for these sequences as for the K and E in EPL001’s KEFNNI. Using the letters given in Figure 6, EPL001’s K7-E10: a-b = 3.06, a-c = 3.49. EPL001’s K9-E10: a-b = 11.09, a-c = 12.24. sSgII-9’s K3-E6: a-b = 3.12, a-c = 2.77. sSgII-9’s E6-K7: a-b = 11.58, a-c = 11.92. Interatomic measurements (20 in total) were made of the FNNI in an in silico model of MKPVFNNI (EPL801, Table 1). These were compared with FNNI measurements in EPL001’s 9KEFNNI14 and in free KEFNNI. The FNNI distances for EPL001 and EPL801 are similar, with the KEFNNI results very dissimilar (see Underlying data)15. F-I measurements for EPL001 are as follows (using letters as given in Figure 6), with EPL801 data in parentheses: d-f = 6.79 (6.34), d-g = 5.49 (7.40), e-f = 7.45 (6.90), e-g = 8.45 (8.96). The figures for KEFNNI are: d-f = 10.57, d-g = 13.56, e-f = 12.21, e-g = 14.01. A chi-squared test of all 20 measurements with EPL001 data as expected showed that the FNNI in MKPVFNNI is highly similar to 11FNNI14 in EPL001: Chi2 = 4.33, dof = 19, 99.5% confidence level. The K2-F5 gaps in MKPVFNNI are a-d = 8.91 and a-e = 9.42, taking ‘a’ as the side-chain nitrogen of K2. For comparison, KEFNNI’s figures for K1-F3 are a-d = 8.69 and a-e = 11.14, while EPL001’s K9-F11 gaps are a-d = 11.38 and a-e = 10.37.\n\nMinimized molecular model of the 14mer MKPLTGKVKEFNNI. Carbon = green; nitrogen = blue; oxygen = red; sulphur = yellow. Hydrogen atoms omitted for clarity. Within the putative epitope KEFNNI (LYS 9 – ILE 14) atoms are indicated (a–k) that were used to determine interatomic distances. a = nitrogen in the side-chain of K9; b = delta carbon in E; c = hydroxyl oxygen in the side-chain of E; d = beta carbon in F; e = gamma carbon of F; f = peptide bond carbon of I; g = delta carbon of I; h = gamma carbon of N13; i = nitrogen of the side-chain of N13; j = peptide bond carbon of N12, k = nitrogen of side-chain of N12.\n\n\nDiscussion\n\nThis report describes the unusual situation where the identity is unclear of an endogenous antigen of an antibody raised to a synthetic peptide, itself of problematic sequence. Epitope mapping is being used here to help solve a purification puzzle, the pieces of which are ‘EPL001’, ‘G530’ and ‘SgII’. It has been demonstrated previously1 that a goat polyclonal antiserum (G530) raised to the synthetic peptide MKPLTGKVKEFNNI (EPL001) labels neuroendocrine and other tissues in various mammalian species and that the endogenous antigen likely relates to secretogranin II (SgII). To determine the endogenous epitope at amino acid resolution, the present report uses IHC, after previous immunoblotting showed that the synthetic epitope resides within or comprises KVKEFNNI, EPL001’s C-terminal section1. Conventional epitope mapping techniques involve X-ray crystallography, nuclear magnetic resonance spectrometry, MS, phage display, ELISA and mutagenesis3, with electron cryomicroscopy a recently developed method of revealing the structures of antibody-antigen complexes. MS-based epitope mapping has been reviewed16, with studies involving synthetic peptide antigens ranging from 47 residues down to 14, as here. In the latter case of a 14mer peptide17, antibodies were interrogated via a panel of synthetic peptides, with alanine substitution, using immunoaffinity-MS and, separately, dot blotting and ELISA. Epitope mapping has been reported for a granin, chromogranin A, using ELISA with a panel of overlapping peptides18. The approach used in the present study has been to probe an enigmatic native antigen in situ because of its resistance to purification. A panel of IHC preabsorption peptides included overlapping trimers, plus a series of alanine-substituted C-terminal tetramers. Immunolabelling is described within the walls of mouse, rat and human cerebral blood vessels and in the wall of the mouse small intestine. Although the EPL001 peptide displays anti-proliferative and pro-apoptotic activities in vitro5 and tissue-mass reducing properties in vivo19 (with relevant immunoneutralizations by anti-EPL001 antibodies in vitro1,5 and in vivo19), implying that an endogenous analogue might do likewise, functional aspects are not a concern in the current report. Neither is the import of the histomorphology. Instead, for antigen elucidation, the focus is exclusively on what the anti-EPL001 antibody binds endogenously in a detailed molecular sense.\n\nThe efficacy of preabsorption and the absence of non-specific binding confirm the ostensible specificity of G530. But to what is it specific? The deployment of two-dozen 3–20mer synthetic peptides in competitive preabsorption studies has demonstrated the importance of a C-terminal NNI in blocking G530 labelling. Although a phenylalanine residue adjacent to the NNI sequence does not appear essential for competition, as the NNI trimer preabsorbs G530 on its own, three N-containing tetramers in the form F - - I, including EPL001’s C-terminal FNNI itself, each delivered the semblance of a concentration-response curve, while two tetramers in the forms - - - I and F - - - did not. In a preliminary analysis, the epitope in the mammalian endogenous antigen could be a C-terminal tetramer, FNNI. Granted EPL001’s ovine provenance, there are no full-length proteins with a C-terminal FNNI in that part of the TrEMBL database20 devoted to Ovis aries (personal communication, Chris Mundy, independent bioinformatician, Liverpool, UK). There are three in a total of 26,443 proteins C-terminating in NNI: two uncharacterised proteins (W5Q2R9 and W5QFS6) and sheep ubiquitin-conjugating enzyme (C5IS99). All have similar sequences C-terminating in MNNI and 152/153 residues, double the expected number from the purification campaign. None of these bears any sequence resemblance to EPL001 beyond the possession of C-terminal NNI. The same is true of 80 hits C-terminating in NNI in 499,317 all-species sequences in UniRef5020, though four more terminate in FNNI without otherwise resembling EPL001 to a significant degree. None ends in EPL001’s C-terminal EFNNI. That leaves the possibility of a proteolytically derived peptide, converting a non-C terminal NNI into a C-terminus, but there are no searchable databases of such items.\n\nAmong endogenous antigens, 10% are contiguous in that they involve a sequence of neighbouring amino acids along a protein backbone16. The other 90% are either entirely non-contiguous, comprising non-consecutive amino acids brought together in space by protein folding, or mixed contiguous and non-contiguous. It has been hypothesized that the native antigen of G530 is related to SgII and that it is mixed, perhaps K·E·F·NNI or KE·F·NNI, corresponding to EPL001’s conjectured maximum likely contiguous epitope 9KEFNNI14 (see Introduction). If KEFNNI endogenously were fully contiguous, then the triplets KEF, EFN and FNN might be expected to block the antibody, but they don’t – though FNN extended to FNNA does to a modest extent (Figure 5). That NNI alone among the trimers successfully preabsorbed G530 indicates that NNI is probably the only contiguous epitopic element endogenously. The sole NNI in the SgII parent protein is not preceded by an F (see Table 1, third item, for the relevant sequences of rSgII, hSgII and sSgII). This implies that the endogenous epitope is thus at least minimally mixed, in the form of F·NNI – and that this is why alanine substitution can be used successfully to probe this part of the epitope. Epitopes cannot be predicted reliably from amino acid sequences, according to a survey of MS epitope mapping, with structure-based rules lacking16. This review found 57 relevant papers from 1986–2015, disclosing 63 epitopes. These ranged in size from 4–71 amino acid residues, with a mean of 15, median of 12 and mode of 8. The present epitope might be F·NNI, but smallness renders this unlikely. That free FNNI is markedly less preabsorptive than EPL001 supports the view that there is more to the epitope than F·NNI.\n\n‘SgII relatedness’ arose on the basis of the present antibody’s deployment in an antigen-capture campaign directed at rat hypothalamus aqueous extract1. SgII itself has been described previously in secretory granules in human astrocytes21 and in mouse cerebellar brush cells22, as well as in rat lateral hypothalamic neurons23,24 and endocrine cells of the small intestine25. The tissue distribution of SgII, however, does not match that of the G530 binding site (as reported here and discussed previously1). The G530 immunopurification campaign did not bring forth SgII-70 as such, but a larger protein identified by the MS software as Q8CGL8, a splice variant of rSgII1. This item has a single non-C terminal NNI that the present work suggests would not be seen by the antibody. It can, however, be surmised that such an NNI might be seen in the presence of other relevant co-located non-contiguous epitope residues, notably F. This is perhaps why FNNA is preabsorptive. Western blots with G530 on sheep serum and rat PC12 conditioned medium visualized single bands at ~7+ kDa (op. cit.). These monobands related to extracellular secreted entities. In contrast, rat hypothalamus yielded three or more close bands around 7+ kDa, whether the aqueous extract was subjected to anion exchange chromatography or purified using a G530 affinity column1. Staining intensity increased down the gels, with all bands preabsorbed by EPL001. The hypothalamic bands represent intracellular forms. They could be intermediates in the processing of SgII-70 towards secretion. This suggests that a non-C-terminal NNI becomes C-terminal, with the IHC exhibiting pre-SgII-70 as well as SgII-70. In this model, G530 is monoepitopic in both senses, towards the synthetic antigen and the endogenous antigen, but in the latter case there is more than one (appropriately folded) SgII-related form.\n\nG530 labels features within the walls of the cerebrovasculature, labelling which is likely to represent at least in part the smooth muscle cell layer. This interpretation is strongly supported by the association of this labelling with that for LRP1, a marker for smooth muscle cells26. No association has been reported between SgII and vascular smooth muscle cells, although secretoneurin (SN), a 33mer peptide derived from the proteolytic processing of SgII has angiogenic properties27. The SN sequence does not contain NNI and has no overlap with that of EPL001. Another SgII peptide is EM6628. This 66mer does possess SgII’s NNI but the sequence of EM66 otherwise does not resemble that of EPL001 and includes no F. If a peptide, possibly with a C-terminal NNI, is processed from SgII then it must be derived via a different proteolytic pathway than SN or EM66.\n\nImmunostaining was paradoxically enhanced by ANNI (Figure 5 and Table 1, where ANNI is recorded as the NNI sequence in hSgII and sSgII). Binding of this tetramer to tissue can be suspected, via its alanine N terminus, providing additional NNI epitopes for the antibody to bind. The 14mer peptide EPL143 was preabsorptive (Table 1). In this case a culminating NNI is preceded by K, showing that G530 may be able to recognize any C-terminal NNI, though the F tetramer data indicate that this is not the endogenous epitope in full. Molecular modelling upheld the immunosorbent trimer NNI as a passable representation in space of EPL001’s 12NNI14, although the likeness narrowly escaped statistical significance. Referring to three peptides in particular, the spatial resemblance in each case to EPL001’s 11FNNI14 is FNNI = MKPVFNNI (both significantly associated)>KEFNNI (NS). In contrast, the preabsorption power ranking is FNNI>KEFNNI>MKPVFNNI. The activity of KEFNNI supports the relevance of KE to the endogenous epitope, in addition to FNNI. (The relative weakness and variability of KEFNNI as a preabsorptive agent and the divergent dimensions of the hexamer from the parent peptide made alanine substitution of the hexamer an unpromising option.) MKPVFNNI, with lower immunosorbence, lacks an E. This indicates that E is a key component in the endogenous epitope, especially as the K-F gaps are similar in KEFNNI and MKPVFNNI.\n\nOvine SgII-9 is MLKTGEKPV (see Introduction), while human SgII-9 has one difference, involving a dissimilar type of amino acid: MLKTGEKPN. As IHC staining is seen in tissue sections from both species1, the V in sSgII-9 and hence in EPL001’s C-terminal section (7KVKEFNNI14) is arguably irrelevant to the epitope. Side-chain interatomic distances (Figure 6: a–b and a–c) between K and E in sSgII-9 (MLKTGEKPV) are strikingly smaller, at ~3 Å, than those relating to the KE in EPL001 (MKPLTGKVKEFNNI), but those of EK in sSgII-9 (MLKTGEKPV), at a little under 12 Å, are similar to those of EPL001’s 9KE10. Leaving aside any contribution to the epitope of nearby peptide backbone atoms and potential reverse-sequence steric differences, this first-order fit supports the deductions that the synthetic epitope of the G530 antibody is EPL001’s 9KEFNNI14 (Figure 6, LYS 9 – ILE 14) and that the endogenous epitope is KE·F·NNI. This latter refines an earlier prediction1 of K·E·F·NNI. By species, the SgII-related epitopes are proposed to be: sheep (W5QEU8), 373KE372·?F·236NNI238; human (P13521), 374KE373·?F·238NNI240; rat (P10362), 376KE375·?F·239NNI241; and mouse (Q03517), 376KE375·?F·239NNI241.\n\nThe foregoing is consistent with there being a peptide derivative of SgII of ~70 residues that N-terminates in MLKTGEKPV/N and C-terminates in NNI, with these motifs sufficiently close together in space to be seen by an antibody. This is a piquant deduction because of residue numbering, which in sSgII is as follows: 367MLKTGEKPV375 and 236NNI238. Reverse peptide splicing is implied by the present results or splicing from separate SgII molecules. Peptide splicing has been reported for another granin protein, chromogranin A, in an SgII-relevant intracellular locus, the secretory vesicle29.\n\nThe immunosorbent power of EPL001 – against which peptide the G530 antibody was of course raised – overtops that of any of its C-terminal components in isolated form. The full 14mer alone seems to present the relevant residues in an appropriate consecutive approximation of a mixed endogenous epitope, for full binding. The relationship between the bioinformatically obscure EPL001 sequence and the proposed SgII-related endogenous antigen is in fact a circular conundrum: how can an endogenous protein be encoded in a synthetic peptide in such a way that an antibody to the synthetic peptide can get back to the endogenous protein? A speculative solution to this is as follows: faced with sSgII-70 the Edman machine did not provide a faithful N-terminal sequence (except for the initial methionine). Instead, it read available superficial residues, of the sort recognized indeed by antibodies. EPL001 thus represents epitope mapping by aberrant Edman sequencing. Hence an anti-EPL001 antibody recognizes sSgII-70. The reason that Edman sequencing was befuddled is deduced to be sSgII-70’s structure (relating perhaps to sorting domain chemistry), which lends itself to depolymerisation on the machine’s analytical matrix, a subject for further work.\n\nProbing, via IHC preabsorption, an endogenous epitope that might be non-contiguous using a panel of short synthetic peptides, while requiring careful interpretation and a guiding hypothesis, has proved productive. A key insight is that antibody binding can be blocked with less than a full complement of epitope residues. Within the EPL001 14mer peptide MKPLTGKVKEFNNI, the epitope of the anti-EPL001 G530 antibody is evidently 9KEFNNI14. This must be so, as the endogenous epitope is deemed to be KE·F·NNI, a mixed contiguous and non-contiguous antibody binding site, as predicted by the hypothesis of antigen relatedness to SgII. The present data are thus consistent with the hypothesis that the anti-EPL001 antibody binds to an SgII related epitope. The postulated SgII-70 evidently N-terminates in MLKTGEKPV/N and C-terminates in NNI. The next desideratum, en route to hormone substantiation, is a full primary sequence.\n\n\nData availability\n\nFigshare: Immunohistochemical labelling within the walls of blood vessels in the human cingulate cortex (BA24) with antibody G530. https://doi.org/10.6084/m9.figshare.9885536.v16.\n\nThis project contains raw images from Figure 1.\n\nFigshare: Human visual cortex labelled with G530 antibody or a commercial antibody to LRP1. https://doi.org/10.6084/m9.figshare.9879719.v17.\n\nThis project contains raw images from Figure 2.\n\nFigshare: Immunohistochemical labelling by antibody G530: blocking with cognate peptide. https://doi.org/10.6084/m9.figshare.9879734.v18.\n\nThis project contains raw images form Figure 3\n\nFigshare: Immunohistochemical labelling of mouse small intestine by antibody G530. https://doi.org/10.6084/m9.figshare.9879773.v19.\n\nThis project contains raw images form Figure 4.\n\nFigshare: Immunohistochemistry with antibody G530 - tittering with tetramers. https://doi.org/10.6084/m9.figshare.988499610.\n\nThis project contains raw images form Figure 5.\n\nFigshare: Immunohistochemical labelling of mouse small intestine and rat brain by antibody G530 - Table data. https://doi.org/10.6084/m9.figshare.9884363.v111.\n\nFigshare: Immunohistochemical labelling of mouse small intestine by antibody G530 - competition by tripeptides. https://doi.org/10.6084/m9.figshare.9884447.v112.\n\nFigshare: Immunohistochemical labelling of mouse small intestine by antibody G530 - competition by tetramers. https://doi.org/10.6084/m9.figshare.9884534.v113.\n\nThe above three projects contain raw images behind Table 1.\n\nFigshare: Immunohistochemical labelling of blood vessels within human visual cortex (BA17) and in mouse small intestine by antibody G530 - competition by KEFNNI and MKPVFNNI. https://doi.org/10.6084/m9.figshare.9884624.v114.\n\nThis project contains raw data discussed in the Results section.\n\nNewton, Russell (2019): Interatomic Distances Chi-Squared Test. figshare. Dataset. https://doi.org/10.6084/m9.figshare.9913040.v115.\n\nThis project contains inter-atomic distances for peptide sequences assessed in the study.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Author contributions\n\nJEH conceived the hypothesis; IJC raised the antiserum; experiments were designed by DRH and JEH; DRH conducted the studies, generating and analysing the data; RPN performed the molecular modelling in silico; JEH and DRH wrote the paper, with co-authorial input; all authors approved the work for publication.\n\n\nAcknowledgments\n\nThe members of Endocrine’s Scientific Advisory Board are thanked, especially for support on bioinformatics (Chris Mundy), logistics (Dave Copsey) and statistics.\n\n\nReferences\n\nHart JE, Clarke IJ, Risbridger GP, et al.: Mysterious inhibitory cell regulator investigated and found likely to be secretogranin II related. PeerJ. 2017; 5: e3833. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHart JE: The body has a brake: micrin is a postulated new gonadal hormone curbing tissue overgrowth and restricting reproduction. Med Hypotheses. 2014; 83(6): 775–86. PubMed Abstract \| Publisher Full Text\n\nCourel M, Vasquez MS, Hook VY, et al.: Sorting of the neuroendocrine secretory protein Secretogranin II into the regulated secretory pathway: role of N- and C-terminal alpha-helical domains. J Biol Chem. 2008; 283(17): 11807–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbbott WM, Damschroder MM, Lowe DC: Current approaches to fine mapping of antigen-antibody interactions. Immunology. 2014; 142(4): 526–35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHart JE: Proteinaceous compounds. US patent 8367801 granted to Endocrine Pharmaceuticals Ltd 2013; European patent EP 2234632 granted 2014. Reference Source\n\nHowlett D: Immunohistochemical labelling within the walls of blood vessels in the human cingulate cortex (BA24) with antibody G530. figshare. Figure. 2019. http://www.doi.org/10.6084/m9.figshare.9885536.v1\n\nHowlett D: Human visual cortex labelled with G530 antibody or a commercial antibody to LRP1. figshare. Figure. 2019. http://www.doi.org/10.6084/m9.figshare.9879719.v1\n\nHowlett D: Immunohistochemical labelling by antibody G530: blocking with cognate peptide. figshare. Figure. 2019. http://www.doi.org/10.6084/m9.figshare.9879734.v1\n\nHowlett D: Immunohistochemical labelling of mouse small intestine by antibody G530. figshare. Figure. 2019. http://www.doi.org/10.6084/m9.figshare.9879773.v1\n\nHowlett D: Immunohistochemistry with antibody G530 - tittering with tetramers. figshare. Figure. 2019. http://www.doi.org/10.6084/m9.figshare.9884996\n\nHowlett D: Immunohistochemical labelling of mouse small intestine and rat brain by antibody G530 - Table data. figshare. Figure. 2019. http://www.doi.org/10.6084/m9.figshare.9884363.v1\n\nHowlett D: Immunohistochemical labelling of mouse small intestine by antibody G530 - competition by tripeptides. figshare. Figure. 2019. http://www.doi.org/10.6084/m9.figshare.9884447.v1\n\nHowlett D: Immunohistochemical labelling of mouse small intestine by antibody G530 - competition by tetramers. figshare. Figure. 2019. http://www.doi.org/10.6084/m9.figshare.9884534.v1\n\nHowlett D: Immunohistochemical labelling of blood vessels within human visual cortex (BA17) and in mouse small intestine by antibody G530 - competition by KEFNNI and MKPVFNNI. figshare. Figure. 2019. http://www.doi.org/10.6084/m9.figshare.9884624.v1\n\nNewton R: Interatomic Distances Chi-Squared Test. figshare. Figure. 2019. http://www.doi.org/10.6084/m9.figshare.9913040.v1\n\nOpuni KFM, Al-Majdoub M, Yefremova Y, et al.: Mass spectrometric epitope mapping. Mass Spectrom Rev. 2018; 37(2): 229–41. PubMed Abstract \| Publisher Full Text\n\nPetre BA, Drăguşanu M, Przybylski M: Molecular Recognition Specificity of anti-3-nitrotyrosine Antibodies Revealed by Affinity-Mass Spectrometry and Immunoanalytical Methods. In: Popescu C., Zamfir A.D., Dinca N. (eds) Applications of Mass Spectrometry in Life Safety. NATO Science for Peace and Security Series A: Chemistry and Biology. Springer, Dordrecht. 2008; 55–67. Publisher Full Text\n\nCorti A, Longhi R, Gasparri A, et al.: Antigenic regions of human chromogranin A and their topographic relationships with structural/functional domains. Eur J Biochem. 1996; 235(1–2): 275–80. PubMed Abstract \| Publisher Full Text\n\nHaylor JL, Parker E, Risbridger GP, et al.: Inhibition of compensatory renal growth by the N-terminus of a sheep-derived peptide. Regul Pept. 2009; 152(1–3): 48–53. PubMed Abstract \| Publisher Full Text\n\nTrEMBL & UniRef50. Accessed 31 December 2018. Reference Source\n\nHur YS, Kim KD, Paek SH, et al.: Evidence for the existence of secretory granule (dense-core vesicle)-based inositol 1,4,5-trisphosphate-dependent Ca2+ signaling system in astrocytes. PLoS One. 2010; 5(8): e11973. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNunzi MG, Mugnaini E: Aspects of the neuroendocrine cerebellum: expression of secretogranin II, chromogranin A and chromogranin B in mouse cerebellar unipolar brush cells. Neuroscience. 2009; 162(3): 673–87. PubMed Abstract \| Publisher Full Text\n\nBayer L, Mairet-Coello G, Risold PY, et al.: Orexin/hypocretin neurons: chemical phenotype and possible interactions with melanin-concentrating hormone neurons. Regul Pept. 2002; 104(1–3): 33–9. PubMed Abstract \| Publisher Full Text\n\nHervé C, Colard C, Grillon S, et al.: Polyethylene glycol-induced hypovolemia affects the expression of MCH mRNA, but not dynorphin or secretogranin II mRNAs, in the rat lateral hypothalamus. Neurosci Lett. 1998; 248(2): 133–7. PubMed Abstract \| Publisher Full Text\n\nMontero-Hadjadje M, Vaingankar S, Elias S, et al.: Chromogranins A and B and secretogranin II: evolutionary and functional aspects. Acta Physiol (Oxf). 2008; 192(2): 309–24. PubMed Abstract \| Publisher Full Text\n\nBoucher P, Gotthardt M, Li WP, et al.: LRP: role in vascular wall integrity and protection from atherosclerosis. Science. 2003; 300(5617): 329–32. PubMed Abstract \| Publisher Full Text\n\nKirchmair R, Hogue-Angeletti R, Gutierrez J, et al.: Secretoneurin--a neuropeptide generated in brain, adrenal medulla and other endocrine tissues by proteolytic processing of secretogranin II (chromogranin C). Neuroscience. 1993; 53(2): 359–65. PubMed Abstract \| Publisher Full Text\n\nMontero-Hadjadje M, Pelletier G, Yon L, et al.: Biochemical characterization and immunocytochemical localization of EM66, a novel peptide derived from secretogranin II, in the rat pituitary and adrenal glands. J Histochem Cytochem. 2016; 51(8): 1083–95. PubMed Abstract \| Publisher Full Text\n\nDelong T, Wiles TA, Baker RL, et al.: Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion. Science. 2016; 351(6274): 711–714. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "54922", "date": "23 Oct 2019", "name": "Michael O. Glocker", "expertise": [ "Reviewer Expertise Mass spectrometry", "proteome research", "protein structure and function analysis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the manuscript by Howlett et al. the authors report on their findings and thoughts on two main topics: (i) attempts to identify the endogenous antigen protein(s) which is (are) decorated by a polyclonal antibody (pAb), named G530, and through which are visualized e.g. certain blood vessel structures in tissues from different origin by IHC, and (ii) investigations targeted on defining the dominating epitope structure, starting with the amino acid sequence “MKPLTGKVKEFNNI”, named EPL001, which was taken to raise G530 and for which IHC is used as readout.\n\nThe authors' research subjects are of high interest for the scientific community and stand in context with developing and applying state-of-the-art methods for elucidating antibody epitopes which is a necessity to understand scope and limitations of the applicability of antibodies as sophisticated research tools, as diagnostic tools, and so on.\n\nThe authors' work underlying hypothesis seems to be: “Once the antibody’s target structure (which in effect is the antigen’s epitope structure) has been identified, the “endogenous” antigen(s) is (are) identified as well”.\n\nWhile this hypothesis seems logical and following it straight forward for successful antigen identification, the authors face the problem that the amino acid sequence stretch “MKPLTGKVKEFNNI” - which previously had been derived from Edman sequencing - cannot be matched to any known protein sequence of any of the species whose tissues had been investigated by IHC. Unfortunately, neither in this nor in the authors’ previous manuscript on the subject was the Edman sequencing result confirmed (see ref. 1 of Howlett et al.1). Instead the authors admit indirectly that a rather poorly defined protein source had been applied for Edman sequencing (quote: “…but scant amino acid sequence data could be obtained before the target molecule was lost to view.”).\n\nIn an attempt to overcome this shortcoming the authors speculate that the experimentally determined amino acid sequence “MKPLTGKVKEFNNI” might originate from a protein’s partial peptide which was produced by peptide splicing. The idea to assume peptide splicing as a cause of the determined amino acid sequence is driven by (rather weak) amino acid sequence similarities which seemed to point to SgII (or SgII-70) as a potential source of the amino acid sequence in question. Unfortunately, the authors do not provide evidence that peptide splicing should occur with SgII as substrate within the tissues which had been investigated by IHC.\n\nSgII was taken into consideration by the authors because this protein had been listed as potentially identified by IP followed by LC-MS analysis, as is stated. The authors report that in this particular case SgII identification was based on a single peptide match - out of a protein that in case of coming from Drosophila contains 1220 amino acids - when setting an FDR of 5%. Following suggested standards (see Carr et al. 2004; Mol. Cell. Proteom. 3, 531-533, 20042) this identification result would rather be considered questionable. Of even more importance, finding a potential target protein by IP followed by LC-MS cannot replace precise characterization of an antigen’s total amino acid sequence prior to performing epitope mapping experiments. For determining an unknown amino acid sequence on the protein level, see e.g. Yefremova et al. J. Am. Soc. Mass Spectrom. (2015) 26:482-4923.\n\nNext, instead of repeating and/or improving antigen identification upon IP (or by other means) and despite not having unequivocally characterized the assumed antigen’s amino acid sequence, the authors had raised a polyclonal antibody, G530, against a synthetic peptide, named EPL001, which comprises the amino acid sequence “MKPLTGKVKEFNNI”. The authors show that (i) G530 recognizes certain blood vessel structures in tissues from different origin by IHC and (ii) G530-dependent IHC staining can be abolished by blocking G530 upon pre-incubation with EPL001.\n\nEncouraged by the antibody-related IHC staining pattern, the authors herewith justify their epitope mapping experiments which are described in this manuscript, despite the fact that their first try failed to identify the “endogenous” antigen by deducing its identity from its assumed epitope amino acid sequence “MKPLTGKVKEFNNI”.\n\nEpitope mapping with IHC as readout, as conducted in this manuscript, looks like an interesting alternative to other epitope mapping methods and starts with subsequently exposing the antibody of interest, in this case G530, to various peptides which do or do not show binding to the antibody. In this study peptide EPL001 and some derivatives therefrom were applied for pre-incubating G530 prior to conducting IHC staining experiments. However, one has to keep in mind that lack of IHC staining of the investigated tissue sections - which stands for saturation of the antibody’s paratope by peptide binding - is at best an indirect manner of epitope mapping and without appropriate controls lacks proof that loss of IHC staining is not caused by unrelated means, such as addition of detergents, pH change, etc. Unfortunately, the manuscript’s Experimental section does not provide enough information to estimate possible influence of such potential confounding factors. The authors are asked to provide more experimental details (see recommendations in the article guidelines: “Methods sections should provide sufficient details of the materials and methods used so that the work can be repeated by others.”). Also to be considered, binding of the peptide(s) under study to the antibody of interest is not shown directly by this method.\n\nNevertheless, the authors performed the respective blocking experiments with various peptides, which are summarized in table 1 of this manuscript, and report that there are shorter partial peptide structures - with resemblances to EPL001 - which render negative IHC staining, hence block G530. From these results a “motif” of six amino acid residues (“KEFNNI”) is deduced by the authors as being necessary for binding G530 with both, the EPL001 peptide and the as of yet still unknown “endogenous” antigen. While the authors’ reasoning can be accepted for EPL001 and its shorter peptide derivatives, demanding that the “KEFNNI” motif must be present on the “endogenous” antigen of G530 is not automatically warranted.\n\nMoreover, one has to consider that the “KEFNNI” motif is precisely part of, but shorter than, the EPL001 peptide amino acid sequence and, therefore, adds no new information beyond what had been shown by dot blot experiments (contained in ref. 1, Figure 2). Consequently, the authors see themselves forced to narrow their base of their hypothesis on an even shorter piece of amino acid sequence as compared to that of EPL001, their first try with searching for the “endogenous” antigen using an amino acid sequence motif. In other words, the authors loosen stringency for data base search to find the mutual antigen and (as might have been expected) fail again in their attempt to convincingly identify the “endogenous” antigen of G530 by applying their “epitope amino acid sequence-based” strategy with focus on SgII as the potential “endogenous” target.\n\nIn their attempts to provide more evidence on their reasoning the authors include results from molecular modelling approaches by which they intend to substantiate their assumptions about SgII being the “endogenous” antigen and to describe molecular structural features of EPL001 which might be required for antibody binding. Yet, these in-silico investigations remain theoretical and descriptive, hence, they ultimately stay inconclusive and are not convincing with respect to now “nailing” SgII as the “endogenous” antigen.\n\nIntriguingly, throughout this manuscript the authors apply methods whose data are to be interpreted rather indirectly in order to prove or falsify their hypothesis instead of using methods whose data provide results which can be directly interpreted to come to unequivocal conclusions. In other words, the authors try to compensate lacking experimental evidence with unproven theories. One is missing experiments which (i) deliver direct evidence about the nature of the epitope’s amino acid sequence(s) and (ii) allow determining the identity of the “endogenous” antigen. These circumstances are addressed by the authors in the discussion and outlook of this manuscript but their respective statements remain sketchy.\n\nMore precise outlines about how the authors plan to continue with their attempts to experimentally determine the “endogenous” antigen of G530 ought to be added to this paragraph of the manuscript. The authors could mention that despite their first unsuccessful attempts with “aqueous” protein extracts it might seem more promising to retrieve the full length antigen protein, e.g. by immunoprecipitation, with protein extracts which also contain less soluble proteins (see e.g. DeCaprio and Kohl, Cold Spring Harb Protoc 2017 doi: 10.1101/pdb.prot0985664). They could mention that they intended to perform an in-depth characterization of the pulled-down and confirmed antigen protein, e.g. by mass spectrometric methods (see e.g. Yefremova et al. J. Am. Soc. Mass Spectrom. (2015) 26:482-4923). With respect to the epitope mapping and antibody recognition motif search, the authors could point to next apply methods which are capable to directly show binding to antibodies of peptides with varying amino acid sequences. A mass spectrometry-based method which is capable to do this is named “ITEM-THREE” and has recently been published by us (see Danquah et al. Mol. Cell. Proteom. (2019) 18:1543-15555).\nMinor comments:\n\nThe term “primary sequence” ought to be deleted from the manuscript and replaced by either “amino acid sequence” or “primary structure”, depending on what of the two is to be described.\n\nThe M+M section needs more precise descriptions so that the “storage conditions” of the peptides can be understood. The pre-incubation experiments need to be described in more detail. Buffer compositions, protein and peptide concentrations, and pH need to be given.\n\nHow was the antiSGII antibody performance tested? Please add details. Without knowing whether the antibody is in fact capable of binding to SgII it is difficult to estimate the mentioned IHC results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5043", "date": "05 Dec 2019", "name": "D Howlett", "role": "Author Response", "response": "We are honoured to receive Professor Glocker’s extensive analysis.1. EPL001. The EPL001 sequence has been a deep source of perplexity. As described in Introduction it was seen once unambiguously during physicochemical purification and three times less fully, with appropriate bioactivity and MALDI MS correlates. Synthesized as a 14mer peptide EPL001 surprised by displaying anti-organotrophic and reproductive effects, as described in Introduction and Discussion, in line with the hypothesis underlying the entire project, of the existence of a tissue-mass reducing reproductively related hormone. Anti-EPL001 antibodies provided images of neuroendocrine relevance and immunoneutralised relevant bioactivities. Even though it is bioinformatically obscure, EPL001 is impossible to ignore, in our view: it is telling us something.2. Potential use of MS in epitope mapping. The project experience with MS has been productive overall but problematic. We chose IHC for epitope mapping because we felt there was a reasonable probability of success, even though IHC is not a recognised route to defining antibody binding. The probability of success in MS-enabled epitope mapping, which in the case Professor Glocker’s outstanding method features a proteolytic enzyme, is more conjectural, as the history of the use of MS in the present project indicates, particularly in regard to the curious use of formalin fixation. The sheep plasma fraction from which the Edman EPL001 sequence was obtained yielded a peak in MALDI-TOF MS at m/z 7583.64 (Fig. 2 in Hart JE, 2013. Proteinaceous compounds: US patent 8367801, also EP 2234632, 2014; available at https://www.google.com/patents/US8367801?dq=Proteinaceous+compounds.&cl=en). Small peaks like this could be obtained in MALDI by hitting samples hard with the laser. Electrospray ionisation yielded nothing. The use of proteolysis with multiple MS modalities (see new section in Introduction) was similarly fruitless. Orbitrap LC-MS with trypsinisation eventually took the project to ‘SgII relatedness’, which evaluation cohered with the evidence from IHC, purification, westerns and so on, including a fruit fly candidate antigen Q9W2X8 that itself unexpectedly turned out to be granin-like. (MS IDs were on the basis of sole tryptic peptides, granted, but the mammalian and fruit fly proteins each had homologues of the other’s identifier, as newly described in an extended section of Introduction, involving new statistics and bioinformatics.) This apparent SgII advance was on the basis of formalin crosslinked feedstock in an immunoprecipitation campaign featuring aqueous extract of rat hypothalamus. Frozen unfixed material provided no credible hits. Formalin fixation with antigen retrieval had been learnt from IHC. We have found no precedents for the use of formalin in factor hunt purification and MS. The need for crosslinking presumably speaks of factor lability. Notably, though, the sought-for 70mer was not found. Instead a larger SgII entity was identified, putatively a processed intermediate. More important than further epitope work now is a full amino acid sequence.3. The connection with SgII. The IHC images in reference 1 are granin-like. They could be taken straight out of a paper on one of the SgII derived peptides. The granin suspicion starts with IHC, not with purification MS or bioinformatics. Referring to bioinformatics, there are two kinds in the paper: the existing material is in Introduction and refers to the second sorting domain of SgII. The bioinformatics in Discussion has been subject to significant augmentation on the basis of ‘NNI-omics’. The NNI-ome is that part of the ovine proteome comprising proteins with at least one example of the motif NNI, the final triplet in EPL001 important in the epitope of the anti-EPL001 antibody G530. The sift reaches SgII as the lead candidate. In Discussion there is a new pen-ultimate paragraph. This in effect describes a sequence alignment between EPL001, sSgII and the fly protein Q9W2X8 as either meaningful or a remarkably freakish coincidence. In our view, the sought-for factor is very likely to be secretogranin II related.Although an interim report following the 2017 paper (reference 1), the present account is proffered as a paper of record, which colleagues will need in efforts at confirmation or refutation. It might also be of interest to the epitope mapping community.Professor Glocker is thanked for stimulating many improvements to our paper." } ] }, { "id": "54975", "date": "24 Oct 2019", "name": "Steven D. Shnyder", "expertise": [ "Reviewer Expertise Tumour cell biology", "proteomics", "preclinical cancer pharmacology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study describes an attempt to clarify which epitope an antibody raised to a candidate peptide EPL001, G530, binds to.\nA rather crude methodology of using IHC with preabsorption of G530 with tripeptides was applied. One would normally expect a couple of techniques to be used to confirm the sequence, such as using a more sophisticated and definitive analytical method such as using MS, whereas in this study they have relied on the presence or absence of immunolabelling on tissue sections, which is rather more subjective. The findings would have been more convincing if backed up using a more sophisticated analytical technique.\nAs it stands I think the study would need to provide further experimental evidence to be worthy of indexing, such as including experiments where functional studies looking at SgII binding/blocking are carried out with G530 along with a known SgII antibody.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5025", "date": "05 Dec 2019", "name": "D Howlett", "role": "Author Response", "response": "We thank Dr Shnyder for his input.The factor under investigation is refractory to MS, a constraint now highlighted in the revised version of the paper.A research impasse called for highly unusual measures: the deployment of epitope mapping in a factor hunt (as spelt out in a new first sentence to Introduction), the use of IHC for epitope mapping (with automated image analysis to eliminate subjectivity), the adoption of rareified bioinformatics (newly uprated, see Discussion in particular), bespoke statistics (also uprated) and molecular modelling.Confirming for Dr Shnyder, a known antibody to SgII was used in the study as a comparator and found to yield a different staining pattern to that of G530. To clarify the situation this sentence has been added to Discussion: ‘So, G530 does not see full-length SgII.’ The paper describes novelty under duress. The pursuit of an elusive factor goes on." } ] } ]	1	https://f1000research.com/articles/8-1732

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